Skip to main content
F1000Research logoLink to F1000Research
. 2022 Mar 4;9:1371. Originally published 2020 Nov 26. [Version 2] doi: 10.12688/f1000research.27657.2

First report of Serratia marcescens associated with black rot of Citrus sinensis fruit , and evaluation of its biological control measures in Bangladesh

Mohammed Faruk Hasan 1, Mohammed Asadul Islam 2, Biswanath Sikdar 2,a
PMCID: PMC8581594  PMID: 34804504

Version Changes

Revised. Amendments from Version 1

In the last version of our article there were some mistake and errors. So, we revised the manuscript according to the reviewers suggestions and comments.

Abstract

Background: The present study was designed to isolate and identify the phyto-pathogen responsible for black rot of Citrus sinensis, and to determine its biological control measures.

Methods: The pathogen was isolated from infected oranges and cultured on Luria-Bertani medium. Gram staining method was used to identify the morphological characteristics of the causal agents of the black rot. Advanced molecular technique was applied to facilitate proper detection of the isolated bacteria. Phylogenetic trees were analyzed using the Neighbor-Joining method. Antimicrobial screening was conducted by disc diffusion method. Antagonistic activity was evaluated by well diffusion method.

Results: Gram staining of the causal agent showed rod shaped, small and pink bacteria. Polymerase chain reaction of the 16S ribosomal RNA gene amplified an approximately 1465 bp product. The nucleotide sequences of the isolated bacterial sample 1 (BS1) and bacterial sample 2 (BS2) had 99.34% and 99.45% similarities with the reference of Serratia marcescens sequence in NCBI GenBank. The obtained sequences were deposited in GenBank. Two isolates showed virulence capability on some fresh fruits, which confirmed the stain detection and Koch’s postulates. Allium sativum extract showed the largest (27.33±1.5 mm) diameter of zone of inhibition against BS1, at 30µg/disc concentration. In the antagonistic assay, Rhizobium leguminosarum showed largest (19±1 mm) zone of inhibition against BS1.

Conclusions: Findings of the current investigations are constructive for identification of causative pathogens in Citrus sinensis black rot disease and their biological control measures.

Keywords: Orange, Black rot, Serratia marcescens, 16S rRNA gene, Biological control

Introduction

Orange, Citrus sinensis (L.) Osbeck is the most common and important species among citrus, belonging to the family of Rutaceae. Orange peel has significant contents of vitamin C, dietary fiber, total polyphenols, carotenoids, limonene and dietary minerals ( Barros et al., 2012). Conventionally, oranges fruits are used to treat disorders like constipation, pains, indigestion, diarrhea, bronchitis, tuberculosis, cough, cold, obesity, menstrual disorders, angina, hypertension, nervousness, downheartedness and pressure ( Milind & Chaturvede, 2012).

Serratia marcescens is an ecologically and genetically diverse pathogenic bacteria species belonging to the family of Enterobacteriaceae ( Besler & Little, 2017). S. marcescens may be present as a contamination on the surface of plants or fruits and enter the tissue via natural pores or sores ( Wang et al., 2015). Previous reports showed that this bacteria is an opportunist pathogen that causes diseases in humans, such as cystitis ( Liu et al., 2004), conjunctivitis ( Hume & Willox, 2004) and others.

Serratia marcescens has been reported as a plant pathogen causing yellow vine disease in Citrullus lanatus and Cucurbita pepo ( Sikora et al., 2012), soft rot in Capsicum annuum ( Gillis et al., 2014), Zea mays whorl rot ( Wang et al., 2015), yellow wilt on Helianthus ( Ignatov et al., 2016), leaf rot in Cannabis sativa ( Schappe et al., 2020) and others. Naturally active plant products are important source of eco-friendly bactericides to control different pathogenic bacteria ( Rahman et al., 2014).

To the best of our knowledge, there is no previous report in Bangladesh on black rot disease of Citrus sinensis fruit, including isolating the pathogen and identifying it through molecular approaches. Moreover, biological control of this pathogen by plants extract and antagonistic microbes has not yet been reported.

Therefore, the objective of this study was to isolate and characterize phytopathogenic bacterial strains responsible for black rot of orange, as well as their controlling techniques using biological agents. The 16S rRNA gene sequence analysis and pathogenicity assays were performed to determine the taxonomic position of the isolated bacterial strains.

Methods

Isolation and purification of bacteria

In 2018 (September-December), eight black rotted orange fruits were collected from RDA market, Rajshahi, Bangladesh. Bacterial pure colonies were isolated and purified from infected fruits as described previously ( Lu & Gross, 2010). Briefly, the infected skin of the fruits were excised with a sterile scalpel and disinfected superficially using 70% alcohol for one minute, sodium hypochlorite for one minute, and finally was rinsed three times in sterile distilled water. The samples were then placed in 100 ml of Luria-Bertani broth medium and incubated at 37°C overnight. Bacteria was streaked onto a fresh LB agar plate and incubated for 18 hours at 37°C. After that, eight different single colonies were picked up by loop and streaked on fresh medium plate to obtained pure culture. Finally, two pure cultures were selected and preserved in 80% glycerol-water solution at -80°C. The two pure cultures were characterized and identified using different approaches, as below.

Morphological characterization

Gram staining and motility of bacteria was conducted as previously described ( Zaoti et al., 2018).

Molecular characterization

The two bacterial isolates were sub-cultured on LB liquid medium and incubated at 30°C for 16 hours with continuous shaking. Genomic DNA was extracted using an automated DNA extractor (Model: Maxwell 16; Promega, USA) and suspended into TE buffer. Afterwards, quantity and quality of the isolated DNA was checked using NanoDrop Spectrophotometer (Model: ND2000; Thermo Scientific, USA).

The isolated bacterial DNA was amplified using the universal bacterial 16S rRNA gene PCR primers (27F 5’-AGAGTTTGATCCTGGCTCAG3’ and 1492R 5’-CGGTTACCTTGTTACGACTT-3’), as previously described ( Srinivasan et al., 2015; Tian et al., 2019); genomic DNA was used as a template. The PCR reaction was performed using the method by Hassan et al. (2018) and Hasan et al. (2020), using hot start green master mix (dNTPs, Buffer, MgCl 2, Taq Pol; Cat: M7432; Promega, USA).

A 20 µl reaction mix was used, containing master mixture 10 µl, T DNA (concentration 25–65 ng/µl) 1 µl, each primer (concentration 10–20 pMol) 1 µl and nuclease free water 7 µl. Thermo-cycling parameters were 95°C for 3 minutes, 32 cycles of 95°C for 30 seconds, 48°C for 30 seconds and 72°C for 90 seconds; a final extension step at 72°C was added for 5 minutes, using a PCR machine (Gene Atlas, Model: G2; Astec, Japan). PCR products were run on 1% agarose gel (Cat: V3125; Promega, USA) and visualized under alpha imager UV trans-illumination (Model: mini; Protein Simple, USA) with 0.5% ethidium bromide solution (Cat: H5041; Promega, USA) in 1xTAE buffer (Cat: V4251; Promega, USA) using a1kb DNA ladder (Cat: G5711; Promega, USA). PCR products were purified from the agarose gel, using SV gel and PCR clean up system (Cat: A9281; Promega, USA). Purified PCR products were sequenced commercially by Sanger sequencing (Apical Scientific, Malaysia). Sequences were used in a search using the NCBI BLAST tool. Obtained sequences were submitted to GenBank (see Data availability) and compared with the GenBank database.

Evolutionary relationships of taxa

A phylogenetic tree was constructed using the Neighbor-Joining method ( Saitou & Nei, 1987) based on the 16S rRNA gene. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches ( Felsenstein, 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method ( Tamura et al., 2004) and are in the units of the number of base substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X ( Kumar et al., 2018).

Pathogenicity assay

Virulence competencies of the isolates were carried out as previously described by Tafinta et al. (2013). First, healthy, fresh, mature malta, lemon, guava and apple fruits were collected from the RDA market, Rajshahi and were sterilized using water and 70% ethanol. The two isolates were grown on LB agar, suspended in autoclaved distilled water at a concentration of 10 8 cells/ml ( Ignatov et al., 2016) and artificially inoculated into malta, lemon, guava and apple fruits. Autoclaved distilled water was treated as a negative control. All the inoculated fruits showed an oversensitive response after 16 hours. To fulfill Koch’s postulates, bacteria were re-isolated from the artificially infected fruits tissue showing symptoms, and characteristics were confirmed based on colony morphology ( Abdellatif et al., 2015) on LB agar along with PCR results.

In vitro antimicrobial screening of the plant extracts

To evaluate antibacterial activities of the isolates, Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava plants were collected from various places (Kajle and Binodpur villages) in Rajshahi district. Plants extraction was performed using the method described by Kader et al. (2018). Different parts of selected plants, bulb of A. sativum and A. cepa; flowers of H. rosa-sinensis; and leaves of O. sanctum, Z. montanum, and P. guajava were cut into small pieces, air-dried, and ground by blender to form a fine powder. The dried powder of the selected plants (100gm of each plant) were rinsed in methanol (500ml) using a conical flask and kept in a shaking incubator for 15 days. The liquid contents were pressed through Markin cloth followed by filtration using Whatman no. 1 filter paper. Obtained filtered liquids were dehydrated in vacuo to leave a blackish and sticky mass. The extracts were preserved in a refrigerator at 4°C using glass vials.

For antibacterial screening, BS1 and BS2 bacterial cultures were inoculated on LB agar plates. Subsequently, discs of filter paper (6 mm in diameter) were saturated with the plant extracts at the concentration of 30 µg/disc and were impregnated on the surface of plates. The plates were incubated at 37°C for 18 hours. Inhibition zones were measured with the help of a millimeter scale.

Antagonistic control

For evaluation of antagonistic effects, five soil bacteria, Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis were used against isolated bacterial strains. Pure cultures of the soil bacteria were kindly provided by Dr. Md. Salah Uddin, Associate Professor and Director, Microbiology Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi, 6205, as the part of collaboration.

To assess the antagonistic effects, agar well diffusion method was used, as previously described ( Valgas et al., 2007). The agar plate’s surface was inoculated by spreading a volume of the microbial inoculum over the entire agar surface. A 6 mm diameter hole was made aseptically with a cork borer. In total, 40 µl/hole bacterial suspension (10 8 CFU/ml) of the isolates were introduced into the well and were incubated at 37°C for 18 hours. The antagonistic soil bacteria were diffused in the LB agar medium that inhibit the growth of two isolated plant pathogens. The test samples were determined by measuring the diameter of zones of inhibition in term of mm with a transparent scale.

Data analysis

All the experiments and test were done triplicate. All data were expressed as the mean value and standard deviation (M ± SD) using Microsoft Excel software 2013.

Results

Morphological characterization of isolates

Single and pure colonies were isolated from the infected fruit grown in LB agar medium. The isolated colonies (BS1 and BS2) were round and pure cultures showed cream and white colonies ( Figure 1A and C, respectively). Gram stained BS1 and BS2 showed small, rod shaped, pink, motile and Gram-negative colonies ( Figure 1B and D).

Figure 1. Naturally infected postharvest sweet orange fruit showing pure culture and morphological phenotypes.

Figure 1.

Isolated bacterial pure colony ( A) BS1 and ( C) BS2; images taken after 18 hours of incubation at 37°C on LB agar. Gram staining of isolated bacterial pure colony ( B) BS1 and ( D) BS2; images taken under the light microscope at 100X magnification, showing as Gram negative.

Molecular characterization of isolated bacteria

Electrophoretic analysis of the DNA isolated from the bacterial strain revealed sharp high molecular weight of the DNA band, which indicated that the DNA was of good quality and suitable for PCR analysis. From the electrophoretic analysis approximately 1465 bp of PCR product was observed while the negative control did not show any product ( Underlying data ( Hasan, 2020b)).

Evolutionary relationships of taxa

The sequences of the total isolates (BS1 and BS2) were compared to Serratia marcescens sequences in GenBank using BLASTN. The partial 16S rRNA gene sequences (accession no. MT912977 and MT912978) were 99.34% and 99.45% ( Figure 2A and B), identical to the reference sequence for S. marcescens strains (ID: AY514434.1) and (ID: JX315621.2), respectively.

Figure 2. Evolutionary relationships of taxa.

Figure 2.

Phylogenetic tree of ( A) BS1 and ( B) BS2. The evolutionary history was inferred using the Neighbor-Joining method ( Saitou & Nei, 1987). This analysis involved 21 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 1494 positions in the final dataset. The scale bar on the rooted tree indicates a 0.50 substitution per nucleotide position.

Pathogenicity assay

The pathogenicity assay showed similar morphological characteristics of Serratia marcescens causing black rot symptoms ( Figure 3A–E). Isolated bacterial DNA from artificially inoculated malta, lemon, guava and apple fruits showed approximately 1465 bp PCR products ( Underlying data ( Hasan, 2020d)).

Figure 3. Naturally infected and artificially inoculated fruit showing symptoms of bacterial black rot.

Figure 3.

( A) post-harvest natural infection, ( B) malta (BS1), ( C) lemon (BS1), ( D) guava (BS2), and ( E) apple (BS2) (artificially inoculated four fruits); images taken 10 days after inoculation.

In vitro antimicrobial screening of the plant extracts

The methanol extract of Allium sativum showed the largest diameter of zone of inhibition (27.33±1.5 mm and 9.17±0.29 mm) against the isolates BS1 and BS2, respectively, at 30 µg/disc concentrations. Zingiber montanum leaf extract didn’t show any antibacterial activity against the isolates BS1 and BS2. Data for all plants are given in Figure 4 and in underlying data ( Hasan, 2020e; Hasan, 2020f; Hasan, 2020g).

Figure 4. Antibacterial activities of Allium sativum, Hibiscus rosa sinensis, Ocimum sanctum, Allium cepa, Zingiper montanum, and Psidium guajava plants extract against the isolates BS1 and BS2.

Figure 4.

Methanol was used as solvent, bacteria was cultured in LB, plated were incubated at 37°C for 18 hours.

Antagonistic activity of soil bacteria against the isolates

The largest inhibition zone was found to be 19±1 mm for Rhizobium leguminosarum followed by 14±1 mm for Rhizobium phaseoli against the isolated bacteria. Brevibacillus borstelensis showed no zone of inhibition against the isolated bacteria (images are given in underlying data ( Hasan, 2020i; Hasan, 2020j)). Data are given in Figure 5.

Figure 5. Antagonistic activity of soil bacteria, Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis against the isolates BS1 and BS2 (it was done in Figure 4).

Figure 5.

Isolates were grown on LB agar; test bacteria were cultured and incubated at 37°C for 18 hours.

Discussion

Morpho-molecular identification has been used as universal adequate procedure to detect bacterium through all key phyta. Evaluation of the bacterial 16S rRNA gene, sequencing has developed as a favored genetic method ( Clarridge, 2004). Now a day, it has also developed as a significant technique to identify an unfamiliar bacterium to the genus or species level ( Sacchi et al., 2002). To distinguish this new disease from the identified rot diseases stated earlier, we propose ‘black rot’ as the common name for the orange disease.

In the present study, the isolated bacterial strains were revealed as gram negative and motile. Tian et al. (2019) reported that Serratia marcescens is a rod-shaped, gram negative bacterium isolated from waste samples which support our present findings. PCR and sequencing was accomplished by 16S rRNA gene amplification for molecular detection of the isolated bacterial strains. Wang et al. (2015) found an identity of 99% to Serratia marcescens strain CPO1 by the BLAST search. Similar result was also reported previously by Altschul et al. (1990). Pathogenicity efficiency of the isolates were confirmed the stains detection using malta, lemon, guava and apple fruits. No significant resistance to this bacterial pathogen was observed in the investigation of the tested fruits. Moreover, Allium sativum plant extract and Rhizobium leguminosarum showed remarkable inhibitory potency against BS1. Villar de Queiroz & Soares de Melo (2006) reported that the strain R3.5 of S. marcescens has an antagonist to P. parasitica in vitro. Present findings revealed the isolates as Serratia marcescens strain, which is responsible for black rot disease of Citrus sinensis fruits.

Conclusions

Bacterial black rot reduces the quantity and quality of the orange and cause chronic losses to the farmers and fruit industries. Advanced molecular approaches along with morphological characterization confirmed the identity of the bacteria as Serratia marcescens. In order to biological control measures of the isolated bacteria, Allium sativum plant extract and Rhizobium leguminosarum bacteria showed promising results. In conclusion, Serratia marcescens causing black rot of orange fruit is a new disease in Bangladesh. Further investigation is needed to better comprehend the biology of the isolates, disease development procedure, and to develop orange varieties resistant or biological control to the bacterial disease in near future.

Data availability

Underlying data

Serratia marcescens strain BS1 containing 16S rRNA gene and partial sequence on GenBank. Accession number, MT912977: https://www.ncbi.nlm.nih.gov/nuccore/MT912977.1/

Serratia marcescens strain BS2 containing 16S rRNA gene and partial sequence on GenBank. Accession number, MT912978: https://www.ncbi.nlm.nih.gov/nuccore/MT912978.1/

Figshare: Naturally infected postharvest sweet orange fruit showing pure culture and morphological phenotypes, https://doi.org/10.6084/m9.figshare.13108142 ( Hasan, 2020a).

This project contains the following underlying data:

  • -

    Original, unedited images as shown in Figure 1

Figshare: PCR amplification of 16S rRNA gene generated from bacteria, https://doi.org/10.6084/m9.figshare.13140074 ( Hasan, 2020b).

This project contains the following underlying data:

  • -

    Original, unedited gel image for BS1 (left lane) and BS2 (right lanes) isolates

Figshare: Naturally infected and artificially inoculated sample fruits showing symptoms of bacterial black rot, https://doi.org/10.6084/m9.figshare.13109120.v3 ( Hasan, 2020c).

This project contains the following underlying data:

  • -

    Original, unedited images as shown in Figure 3

Figshare: PCR amplification of 16S rRNA gene generated from bacteria for virulence test, https://doi.org/10.6084/m9.figshare.13140017 ( Hasan, 2020d).

This project contains the following underlying data:

  • -

    Original, unedited gel images for BS1 isolate, and infected malta, lemon, guava and apple fruits (left to right lanes)

Figshare: Antibacterial activities of methanolic extract of some plant against the isolates BS1 and BS2, https://doi.org/10.6084/m9.figshare.13160348 ( Hasan, 2020e).

Figshare: Antibacterial activities of some plants extract against the isolate BS1, https://doi.org/10.6084/m9.figshare.13160615 ( Hasan, 2020f).

This project contains the following underlying data:

  • -

    Images of zone of inhibition of BS1 for Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava

Figshare: Antibacterial activities of some plants extract against the isolate BS2, https://doi.org/10.6084/m9.figshare.13160648 ( Hasan, 2020g).

This project contains the following underlying data:

  • -

    Images of zone of inhibition of BS2 for Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava

Figshare: Antagonistic activity of soil born bacteria against the isolates BS1 and BS2, https://doi.org/10.6084/m9.figshare.13139981.v1 ( Hasan, 2020h).

Figshare: Antagonistic activity of soil born bacteria against the isolates BS1, https://doi.org/10.6084/m9.figshare.13108055 ( Hasan, 2020i).

This project contains the following underlying data:

  • -

    Images of zone of inhibition of BS1 for Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis.

Figshare: Antagonistic activity of soil born bacteria against the isolate BS2, https://doi.org/10.6084/m9.figshare.13108061 ( Hasan, 2020j).

This project contains the following underlying data:

  • -

    Images of zone of inhibition of BS2 for Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

The authors would like to thank Mr. Md. Roushan Ali, MS Research Fellow, Chinese Academy of Sciences, Kunming, China, for software support to analyses the sequences data.

Funding Statement

This research work was supported by the Ministry of Education, Government of the People's Republic of Bangladesh (2019-2021) [No. 3900.0000.09.06.217/2BS 91/170].

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 2; peer review: 2 approved

References

  1. Abdellatif E, Kaluzna M, Helali F, et al. : First report of citrus bacterial blast and citrus black pit caused by Pseudomonas syringe pv. syringae in Tunisia. New Disease Rep. 2015;32(1):35. 10.5197/j.2044-0588.2015.032.035 [DOI] [Google Scholar]
  2. Altschul SF, Gish W, Miller W, et al. : Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410. 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]
  3. Barros HR, Ferreira TA, Genovese MI: Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem. 2012;134(4):1892–1898. 10.1016/j.foodchem.2012.03.090 [DOI] [PubMed] [Google Scholar]
  4. Besler KR, Little EL: Diversity of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease in Georgia. Plant Dis. 2017;101(1):129–136. 10.1094/PDIS-05-16-0618-RE [DOI] [PubMed] [Google Scholar]
  5. Clarridge JE, 3rd: Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases. Clin Microbiol Rev. 2004;17(4):840–862, table of contents. 10.1128/CMR.17.4.840-862.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Felsenstein J: Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39(4):783–791. 10.1111/j.1558-5646.1985.tb00420.x [DOI] [PubMed] [Google Scholar]
  7. Gillis A, Rodriguez M, Santana M: Serratia marcescens associated with bell pepper ( Capsicum annuum L.) soft-rot disease under greenhouse conditions. Eur J Plant Pathol. 2014;138(1):1–8. 10.1007/s10658-013-0300-x [DOI] [Google Scholar]
  8. Hasan M: Naturally infected postharvest sweet orange fruit showing pure culture and morphological phenotypes. Figshare. 2020a. 10.6084/m9.figshare.13108142 [DOI] [Google Scholar]
  9. Hasan M: PCR amplification of 16S rRNA gene generated from bacteria. Figshare. 2020b. 10.6084/m9.figshare.13140074 [DOI] [Google Scholar]
  10. Hasan M: Naturally infected and artificially inoculated sample fruits showing symptoms of bacterial black rot. Figshare. 2020c. 10.6084/m9.figshare.13109120.v3 [DOI] [Google Scholar]
  11. Hasan M: PCR amplification of 16S rRNA gene generated from bacteria for virulence test. Figshare. 2020d. 10.6084/m9.figshare.13140017 [DOI] [Google Scholar]
  12. Hasan M: Antibacterial activities of methanolic extract of some plant against the isolates BS1 and BS2. Dataset. Figshare. 2020e. 10.6084/m9.figshare.13160348 [DOI] [Google Scholar]
  13. Hasan M: Antibacterial activities of some plants extract against the isolate BS1. Figshare. 2020f. 10.6084/m9.figshare.13160615 [DOI] [Google Scholar]
  14. Hasan M: Antibacterial activities of some plants extract against the isolate BS2. Figshare. 2020g. 10.6084/m9.figshare.13160648 [DOI] [Google Scholar]
  15. Hasan M: Antagonistic activity of soil born bacteria against the isolates BS1 and BS2. Dataset. Figshare. 2020h. 10.6084/m9.figshare.13139981.v1 [DOI] [Google Scholar]
  16. Hasan M: Antagonistic activity of soil born bacteria against the isolates BS1. Figshare. 2020i. 10.6084/m9.figshare.13108055 [DOI] [Google Scholar]
  17. Hasan M: Antagonistic activity of soil born bacteria against the isolate BS2. Figshare. 2020j. 10.6084/m9.figshare.13108061 [DOI] [Google Scholar]
  18. Hasan MF, Islam MA, Sikdar B: First report on molecular identification of Fusarium species causing fruit rot of mandarin ( Citrus reticulata) in Bangladesh [version 1; peer review: 1 approved]. F1000Res. 2020;9:1212. 10.12688/f1000research.26464.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hassan O, Jeon JY, Chang T, et al. : Molecular and morphological characterization of Colletotrichum species in the Colletotrichum gloeosporioides complex associated with persimmon anthracnose in South Korea. Plant Dis. 2018;102(5):1015–24. 10.1094/PDIS-10-17-1564-RE [DOI] [PubMed] [Google Scholar]
  20. Hume EBH, Willox MDP: Emergence of Serratia marcescens as an ocular surface pathogen. Arch Soc Esp Oftalmol. 2004;79(10):475–481. [PubMed] [Google Scholar]
  21. Ignatov AN, Khodykina MV, Polityko VA, et al. : First report of Serratia marcescens causing yellow wilt disease on sunflower in Russia. New Disease Reports. 2016;33:8. 10.5197/j.2044-0588.2016.033.008 [DOI] [Google Scholar]
  22. Kader SMA, Hasan M, Ahmed S, et al. : Antioxidant, Antibacterial and Cytotoxic activities of Ethanol extract and its different fractions of Sterculia cordata leaves. Discovery Phytomedicine. 2018;5(3):26–33. 10.15562/phytomedicine.2018.64 [DOI] [Google Scholar]
  23. Kumar S, Stecher G, Li M, et al. : MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–1549. 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu JW, Hsu YM, Huang YF: Independent prognostic factors for fatality in patients with urinary tract infection caused by Serratia marcescens. Infect Control Hosp Epidemiol. 2004;25(1):80–82. 10.1086/502297 [DOI] [PubMed] [Google Scholar]
  25. Lu SE, Gross DC: Drippy pod of white lupine: a new bacterial disease caused by a pathovar of Brenneria quercina. Plant Dis. 2010;94(12):1431–1440. 10.1094/PDIS-05-10-0365 [DOI] [PubMed] [Google Scholar]
  26. Milind P, Chaturvede D: Orange: Range of benefits. International Research Journal of Pharmacy. 2012;3(7):59–63. Reference Source [Google Scholar]
  27. Rahman A, Islam R, Al-Reza SM, et al. : In vitro control of plant pathogenic Xanthomonas spp. using Poncirus trifoliata Rafin. EXCLI J. 2014;13:1104–1110. [PMC free article] [PubMed] [Google Scholar]
  28. Sacchi CT, Whitney AM, Mayer LW, et al. : Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis. 2002;8(10):1117–1123. 10.3201/eid0810.020391 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Saitou N, Nei M: The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–425. 10.1093/oxfordjournals.molbev.a040454 [DOI] [PubMed] [Google Scholar]
  30. Schappe T, Ritchie DF, Thiessen LD: First Report of Serratia marcescens Causing a Leaf Rot Disease on Industrial Hemp ( Cannabis sativa). Plant Disease. 2020;104(4):1248. 10.1094/PDIS-04-19-0782-PDN [DOI] [Google Scholar]
  31. Sikora EJ, Bruton BD, Wayadande AC, et al. : First Report of the Cucurbit Yellow Vine Disease Caused by Serratia marcescens in Watermelon and Yellow Squash in Alabama. Plant Dis. 2012;96(5):761. 10.1094/PDIS-09-11-0739-PDN [DOI] [PubMed] [Google Scholar]
  32. Srinivasan R, Karaoz U, Volegova M, et al. : Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens. PLoS One. 2015;10(2):e0117617. 10.1371/journal.pone.0117617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tafinta IY, Shehu K, Abdulganiyyu H, et al. : Isolation and Identification of Fungi Associated with the Spoilage of Sweet Orange ( Citrus sinensis) Fruits In Sokoto State. Nigerian Journal of Basic and Applied Science. 2013;21(3):193–6. 10.4314/njbas.v21i3.4 [DOI] [Google Scholar]
  34. Tamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004;101(30):11030–11035. 10.1073/pnas.0404206101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tian C, Zhao J, Zhang Z, et al. : Identification and molecular characterization of Serratia marcescens phages vB_SmaA_2050H1 and vB_SmaM_2050HW. Arch Virol. 2019;164(4):1085–1094. 10.1007/s00705-019-04169-1 [DOI] [PubMed] [Google Scholar]
  36. Valgas C, Souza SMD, Smânia EF, et al. : Screening methods to determine antibacterial activity of natural products. Brazilian J microbial. 2007;38(2):369–380. 10.1590/s1517-83822007000200034 [DOI] [Google Scholar]
  37. Villar de Queiroz BP, Soares de Melo I: Antagonism of Serratia marcescens towards Phytophthora parasitica and its effects in promoting the growth of citrus. Braz J Microbiol. 2006;37:448–450. 10.1590/S1517-83822006000400008 [DOI] [Google Scholar]
  38. Wang X, Bi T, Li X, et al. : First Report of Corn Whorl Rot Caused by Serratia marcescens in China. J Phytopathol. 2015;163(11–12):1059–1063. 10.1111/jph.12366 [DOI] [Google Scholar]
  39. Zaoti Z, Hasan SM, Hossain M, et al. : Biochemical and Molecular Characterization of Bacterial Wilt Disease of Banana and Evaluation of their Antibiotic Sensitivity. Microbiol Res J Int. 2018;22(6):1–10. Reference Source [Google Scholar]
F1000Res. 2022 Mar 7. doi: 10.5256/f1000research.79529.r126312

Reviewer response for version 2

Ciamak Ghazaei 1

All changes and the revision are approved.

Sincerely,

Ciamak Ghazaei

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Microbiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

F1000Res. 2021 Nov 10. doi: 10.5256/f1000research.30572.r98171

Reviewer response for version 1

Ciamak Ghazaei 1

This manuscript does an appropriate demonstrating significant bacterial strain by isolation and identification a phytopathogen. This research is quite an interesting round up of the "First report of Serratia marcescens associated with black rot of  Citrus sinensis fruit...".

Whilst the information presented is generally suitable, the level of some presentation limits the ease of digestion of the content. In general, the manuscript would benefit from the clearing of sentences as this avoids misunderstanding of facts. Also the authors could consider a susceptibility test (Antibiotic resistance) comparison for isolates of  Serratia marcescens which would be more attractive to the readership.

All the queries and concerns are remarked within the manuscript file.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Microbiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

F1000Res. 2021 Nov 15.
Md Faruk Hasan 1

We corrected the manuscript according to the reviewer comments and suggestions. We also deleted and added some words and sentence following reviewer suggestions.

F1000Res. 2021 Apr 15. doi: 10.5256/f1000research.30572.r81642

Reviewer response for version 1

Mapotso Kena 1

The authors mentioned several methods being used for morphological identification, however, only two methods were used and this must be corrected. Reference to "plant extracts" as biological control is wrong and this must be rectified by the authors.

This further affect the study title which should be modified to accomodate plant extracts. 

It is necessary to also explain in detail how inoculation was done for both isolates.

Under " In vitro antimicrobial screening of the plant extracts", the second but last sentence of the last paragraph needs re-phrasing.

Clarity is needed with some figures and results presentation and this has been highlighted in the text. Discussion can be improved with additional reference to existing information on biological control and use of plant extracts in the control of other pos-harvest diseases.

Some suggested minor editorial corrections are highlighted in the text.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Plant Pathology, biological control of plant pathogens, identification and characterization.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

F1000Res. 2021 Nov 15.
Md Faruk Hasan 1

We read through the manuscript and corrected it according to the reviewer comments.

F1000Res. 2021 Feb 19. doi: 10.5256/f1000research.30572.r78843

Reviewer response for version 1

Kator Liamngee 1

The work is clear, accurately presented, and cites current literature.

The study design is not properly presented. There should be a subheading under the in vitro antimicrobial screening of the plant extracts bearing experimental design. The factors in the experiment should be clearly brought out, treatment combinations and the number of units/lots should be clearly mentioned so one can understand the experimental layout/arrangement.

The work is technically sound but details of methods are not sufficient to allow replications. This is shown in the pathogenicity. The author needs to explain in details how the two isolates, BS1 and BS2 grown on LB agar, were artificially inoculated into Malta, lemon, guava, and apple fruits during the pathogenicity assay; and the nature of the data collected.

The concentrations of the plant extracts used in the in vitro antimicrobial screening should be clearly stated with clear descriptions on how the concentrations were arrived at.

There should be an experimental design for the antagonistic control experiment. The factors in the experiment should be spelt out, treatment combinations (if any) should be mentioned and number of units or lots clearly stated to boost understanding of the arrangement or layout of this particular experiment.

Sources of data underlying the results are available. The statistical analysis and its interpretation is appropriate and all conclusions are adequately supported by the results.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Postharvest Biology, Plant pathology, Postharvest pathology, Biocontrol using plant extracts.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

F1000Res. 2021 Feb 26.
Md Faruk Hasan 1

I am approving the reviewer comments and suggestions.

F1000Res. 2021 Nov 15.
Md Faruk Hasan 1

I am approving the reviewer comments and suggestions.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    Underlying data

    Serratia marcescens strain BS1 containing 16S rRNA gene and partial sequence on GenBank. Accession number, MT912977: https://www.ncbi.nlm.nih.gov/nuccore/MT912977.1/

    Serratia marcescens strain BS2 containing 16S rRNA gene and partial sequence on GenBank. Accession number, MT912978: https://www.ncbi.nlm.nih.gov/nuccore/MT912978.1/

    Figshare: Naturally infected postharvest sweet orange fruit showing pure culture and morphological phenotypes, https://doi.org/10.6084/m9.figshare.13108142 ( Hasan, 2020a).

    This project contains the following underlying data:

    • -

      Original, unedited images as shown in Figure 1

    Figshare: PCR amplification of 16S rRNA gene generated from bacteria, https://doi.org/10.6084/m9.figshare.13140074 ( Hasan, 2020b).

    This project contains the following underlying data:

    • -

      Original, unedited gel image for BS1 (left lane) and BS2 (right lanes) isolates

    Figshare: Naturally infected and artificially inoculated sample fruits showing symptoms of bacterial black rot, https://doi.org/10.6084/m9.figshare.13109120.v3 ( Hasan, 2020c).

    This project contains the following underlying data:

    • -

      Original, unedited images as shown in Figure 3

    Figshare: PCR amplification of 16S rRNA gene generated from bacteria for virulence test, https://doi.org/10.6084/m9.figshare.13140017 ( Hasan, 2020d).

    This project contains the following underlying data:

    • -

      Original, unedited gel images for BS1 isolate, and infected malta, lemon, guava and apple fruits (left to right lanes)

    Figshare: Antibacterial activities of methanolic extract of some plant against the isolates BS1 and BS2, https://doi.org/10.6084/m9.figshare.13160348 ( Hasan, 2020e).

    Figshare: Antibacterial activities of some plants extract against the isolate BS1, https://doi.org/10.6084/m9.figshare.13160615 ( Hasan, 2020f).

    This project contains the following underlying data:

    • -

      Images of zone of inhibition of BS1 for Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava

    Figshare: Antibacterial activities of some plants extract against the isolate BS2, https://doi.org/10.6084/m9.figshare.13160648 ( Hasan, 2020g).

    This project contains the following underlying data:

    • -

      Images of zone of inhibition of BS2 for Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava

    Figshare: Antagonistic activity of soil born bacteria against the isolates BS1 and BS2, https://doi.org/10.6084/m9.figshare.13139981.v1 ( Hasan, 2020h).

    Figshare: Antagonistic activity of soil born bacteria against the isolates BS1, https://doi.org/10.6084/m9.figshare.13108055 ( Hasan, 2020i).

    This project contains the following underlying data:

    • -

      Images of zone of inhibition of BS1 for Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis.

    Figshare: Antagonistic activity of soil born bacteria against the isolate BS2, https://doi.org/10.6084/m9.figshare.13108061 ( Hasan, 2020j).

    This project contains the following underlying data:

    • -

      Images of zone of inhibition of BS2 for Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis.

    Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).


    Articles from F1000Research are provided here courtesy of F1000 Research Ltd

    RESOURCES