Figures
Abstract
Jackfruit-bronzing is caused by bacteria Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii), showing symptoms of yellowish-orange to reddish discolouration and rusty specks on pulps and rags of jackfruit. Twenty-eight pure bacterial strains were collected from four different jackfruit outbreak collection areas in Peninsular Malaysia (Jenderam, Maran, Muadzam Shah and Ipoh). Positive P. stewartii subsp. stewartii verification obtained in the study was based on the phenotypic, hypersensitivity, pathogenicity and molecular tests. Multilocus sequence analysis (MLSA) was performed using four housekeeping genes (gyrB, rpoB, atpD and infB) on all 28 bacterial strains. Single gyrB, rpoB, atpD and infB phylogenetic trees analyses revealed the bootstrap value of 99–100% between our bacterial strains with P. stewartii subsp. stewartii reference strains and P. stewartii subsp. indologenes reference strains. On the other hand, phylogenetic tree of the concatenated sequences of the four housekeeping genes revealed that our 28 bacterial strains were more closely related to P. stewartii subsp. stewartii (99% similarities) compared to its close relative P. stewartii subsp. indologenes, although sequence similarity between these two subspecies were up to 100%. All the strains collected from the four collection areas clustered together, pointing to no variation among the bacterial strains. This study improves our understanding and provided new insight on the genetic diversity of P. stewartii subsp. stewartii associated with jackfruit-bronzing in Malaysia.
Citation: Abidin N, Ismail SI, Vadamalai G, Yusof MT, Hakiman M, Karam DS, et al. (2020) Genetic diversity of Pantoea stewartii subspecies stewartii causing jackfruit-bronzing disease in Malaysia. PLoS ONE 15(6): e0234350. https://doi.org/10.1371/journal.pone.0234350
Editor: Rajarshi Gaur, Deen Dayal Upadhyaya Gorakhpur University, INDIA
Received: January 24, 2020; Accepted: May 23, 2020; Published: June 12, 2020
Copyright: © 2020 Abidin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by IPM-UPM (Grant number: 9494600), FRGS (Grant number: 5540090) and SEAMEO SEARCA (https://www.searca.org/). Nuraizat Abidin was the recipient of UPM-SEAMEO SEARCA scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Jackfruit (Artocarpus heterophyllus) comes from the mulberry family (Moraceae) and is believed to originate from the evergreen forest in Western Ghast, India [1]. Jackfruit is an important crop in Malaysian agriculture and recognised as “Nangka” by Malay verbal. The Economic Transfer programme (ETP) implemented by the Government of Malaysia [2] that includes National Key Economic Area (NKEA) states that “Jackfruit is an important driver of economic activities that potentially and directly contributes toward the Malaysia economic growth measurable by the National Gross Income (GNI) indicator” [3]. Under NKEA Agriculture, which includes Entry Point Project number 7 (EPP 7), it focuses on the increased growth of premium fruits and vegetables of which jackfruit is one of the six high-value fruit crops of EPP 7 [4].
Jackfruit industry in Malaysia is currently under threat with a disease named “jackfruit-bronzing”. Examining and keeping inspection records on the status of jackfruit-bronzing is essential to preserve a high quality and to ensure the security of the local food supply of jackfruit. Recently, there has been three reported cases of jackfruit-bronzing in the Philippines [5], Peninsular Malaysia [6] and Mexico [7]. Visually, bronzing disease on jackfruit is symptomless on the external appearance of the fruit, but it shows yellowish-orange to reddish discolouration and rusty specks on the pulps and rags [5–7]. The other visible symptom according to an earlier report on the outbreak of the disease in Malaysia was obvious reddish-brown spots on the pulps [8].
The causal agent, P. stewartii subsp. stewartii (Smith) Mergaert is a plant pathogenic bacterium of the family Enterobacteriaceae [9]. This bacteria is the only pathogen listed in the quarantine pathogen among the genus Pantoea [10]. The European and Mediterranean Plant Protection Organization (EPPO) stated maize as the primary host for P. stewartii subsp. stewartii, which is widely known as Stewart’s bacterial wilt as well as leaf blight of corn, sweetcorn, maize, dent, flint, flour and popcorn plant, indigenous to North America [11,12]. The first case of a plant disease ever reported among the genus Pantoea was Stewart’s wilt of maize, and the discovery led to major losses in crop yield [13,14].
P. stewartii subsp. stewartii can be identified using the primers designed from the 16S-23S ribosomal DNA (rDNA) internal transcribed spacer (ITS) [6,14] to confirm the identity of the bacterial strains. As the pure bacterial strains have been confirmed positive for P. stewartii subsp. stewartii, pathogenicity test is vital to confirm if the bacterium is the causal agent, or this would disprove their status as novel pathogens [9,15]. Multilocus sequence analysis (MLSA) is considered as the novel standard for the systematics in molecular microbiology expected to produce high resolving power results [16]. Based on partial sequence of gyrB (encoding DNA gyrase subunit B), rpoB (encoding RNA polymerase beta subunit), atpD (encoding ATP synthase F1, β-subunit) and infB (encoding Translation initiation factor IF-2) genes (also known as Housekeeping genes), P. stewartii subsp. stewartii is successfully distinguished from another Pantoea genus. So far, the genetic diversity of P. stewartii subsp. stewartii in Peninsular Malaysia has not been investigated.
Losses in jackfruit yields began with continuous fruit bronzing disease occurrences [6]. It leads to loss of jackfruit production and thus considered as a major constrain with big economic impact in Malaysia [6]. Since the first outbreak in 2017, very limited documentations of jackfruit-bronzing disease in Malaysia has been recorded. Hence, detailed status reports of jackfruit-bronzing disease is significantly important since jackfruit remains as one of the most important commercial fruit crops with a high economic value in Malaysia.
In connection with the first report of P. stewartii subsp. stewartii infected jackfruit in 2017 in Muadzam Shah, Peninsular Malaysia, the objectives of the present study were: (i) to verify the bacterial strains associated with bronzing disease of jackfruit in four outbreak plantations in Peninsular Malaysia are P. stewartii subsp. stewartii, using phenotypic and molecular characteristics; and (ii) to elucidate the genetic diversity of P. stewartii subsp. stewartii strains associated with bronzing disease of jackfruit using multilocus sequence analysis (MLSA) approach.
Materials and methods
Sample collection and isolation of bacterial pathogen
A total of 28 diseased jackfruits (Tekam Yellow J33 variety) were collected from outbreak plantation areas in Peninsular Malaysia, comprising of 7 fruits from Jenderam (Selangor), 7 fruits from Maran (Pahang), 7 fruits from Muadzam Shah (Pahang) and 7 fruits from Ipoh (Perak) from September-November 2017 (Table 1). Fruits were collected randomly, with three replicates for each sample per location (total 21 fruits per collection area). The infected fruits were wrapped, put inside sterile plastic bags, labelled and brought to the laboratory for initial diagnosis and isolation of the pathogen. The isolation procedure was conducted not later than 24 h after the collected samples were brought to the laboratory.
Infected samples were excised, cut and rinsed with sterilized distilled water (containing 1% of sodium hypochlorite, Chlorox™) for 3 minutes. The jackfruit pulps were put on King’s B agar medium, sealed properly and incubated in inverted position for 24 to 48 h at 28º C. After incubation, isolated colonies were subcultured until pure colonies of suspected P. stewartii subsp. stewartii strains were obtained. As mentioned by EPPO, the colonies were expected to be lemon to pale-yellow or orange-yellow in colour, flat to convex, transparent with entire edges, and slow to medium growing [9]. For later use, the pure bacterial cultures were grown in nutrient broth medium with 20% (v/v) glycerol and stored at -80°C.
Ethics statement
For collection of fruits, no specific permits or permission were required for these locations/activities. All field research was conducted on privately-owned farms and with owners’ permission. All locations where the fruits were collected did not involve endangered or protected species.
Verification via phenotypic characterization
The positive phenotypic characterization for the verification of P. stewartii subsp. stewartii were performed based on previous studies verification [5,9,17–19] as mentioned on S1 Table. All tests were performed using the freshly grown colonies of P. stewartii subsp. stewartii (24 to 48 h).
Verification via molecular characterization
P. stewartii subsp. stewartii strains were grown on nutrient broth for 24 to 48 h at 28°C. Using commercial genomic DNA isolation kit, (PrestoTM Mini gDNA Bacteria Kit, Geneaid Biotech LTD., Taiwan), all the 28 bacteria strains were extracted following the protocol provided. The polymerase chain reaction (PCR) was performed in a 25 μl reaction mixture, contained 1 μg of extracted total genomic DNA template, 12.5 μl of 2x DreamTaq Red PCR MasterMix (Thermo Scientific Inc., USA), 9.5 μl of DNase-free water and 1 μl of each primer (Apical Scientific, Malaysia). PCR amplification was performed following protocols, in ‘iCycler’ Thermal Cycler (Bio-Rad Laboratories Inc., USA) to amplify the 16S-23S ITS region, using primer pair: ES16 (5’- GCG AAC TTG GCA GAG AT -3’) and ESIG2c (5’- GCG CTT GCG TGT TAT GAG -3’) [14,20].
Hypersensitive assay
Four-week-old Nicotiana benthamiana transplants were used for hypersensitivity assays. Using syringe, 1 ml of 108 CFU/ml of bacterial suspension (as determined by McFarland Standard, BioMérieux, Marcyl'Etoile, France) was inoculated into a fully expanded tobacco leaf (3 leaves per bacterial culture). Leaves infiltrated with sterile distilled water served as a negative control. The greenhouse condition maintained at temperature range from 26°C to 35°C. The plant response for Hypersensitivity reaction (HR) was recorded after 10 to 36 h post-infiltration. Necrosis of the infiltrated tissues was considered as positive HR, and the absence was considered as a negative test. Four replicates were performed for each strain due to any imprecision concern. Two results were expected for this test, as past studies showed P. stewartii subsp. stewartii strains resulted in positive HR and pathogenic [18] or otherwise resulting negative HR, suggesting that the strains are non-pathogenic due to mutation in the hrp gene [5,11,18].
Pathogenicity test
Detached healthy jackfruit pulps (Tekam Yellow J33 variety) were inoculated with 10 mL of 108 CFU/mL (as determined by McFarland Standard, BioMérieux, Marcyl'Etoile, France) of 24 to 48 h pure P. stewartii subsp. stewartii suspensions. Sterile distilled water was inoculated to control healthy jackfruit pulps. 4 replicates were performed per strain. All 28 bacterial strains treatments were put inside sterile petri dish (properly sealed) and incubated at 28 ºC in a controlled chamber. The evaluation of jackfruit-bronzing symptoms was recorded daily up to two weeks of inoculation.
MLSA
All extracted total genomic DNA were used as a template in the PCR amplification using primers as listed on S2 and S3 Tables. The PCR reactions were performed as described under the previous section.
Electrophoresis of PCR products, DNA purification, sequencing, sequence alignment, phylogenetic analyses and dendrogram construction
Separation of PCR products was performed on a prepared 1% agarose gel with FloroSafe DNA stain. Using Mini Sub™ DNA Cell, (Bio-Rad Laboratories Inc., USA), the PCR amplicons was run at 75 V (Power supply model 1000/500, Bio-Rad Laboratories Inc., USA) for 45 min and later visualized and photographed by Alpha Imager System (Alpha-Innotech, Siber-Hegner, UK).
All amplified PCR products were then sent for purification and sequencing (MyTACG Sequencing, Malaysia). All forward and reverse sequences were assembled and analyzed using Bioedit version 7.2.5 [21]. The FASTA format of the gene sequences were subjected to BLASTn (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) search to determine their similarity to nucleotide sequences available in the GenBank database. This was followed by using ClustalW multiple alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/), with manual adjustments. MEGA7 software [22] was used for the construction of phylogenetic trees using the best fit substitution model identified and calculated by the "Find Best Models" tool. Maximum likelihood tree were then generated with bootstraps values of 1000 for each analysis. The available sequences for these genes in genomes of P. stewartii subsp. stewartii were then compared.
All reference strains were obtained from NCBI online database (S4 and S5 Tables). The sequenced genes from the study have been deposited into the NCBI database as mention on S4 and S5 Tables.
Results
Collection of the sample and symptoms of jackfruit-bronzing
From the total of 28 collected jackfruits, all fruits (7 fruits from Jenderam, 7 fruits from Maran, 7 fruits from Muadzam Shah and 7 fruits from Ipoh) showed the symptoms of bronzing disease (Table 2). The outer appearance of the collected fruits were symptomless. Internally, they showed visible jackfruit-bronzing symptoms either yellowish discolouration with bronzing specks or reddish discolouration on the pulps and rags (Fig 1). The symptoms were inconsistent with 17 of the fruits showed yellowish discolouration with bronzning specks and 11 had reddish discolouration appearance (Table 2).
(a) Yellowish discolouration with bronzing specks symptom collected from Muadzam Shah; (b) Yellowish discolouration with bronzing specks symptom collected from Ipoh; (c) Reddish discolouration on the pulps of jackfruit collected from Jenderam; (d) Reddish discolouration on the pulps of jackfruit collected from Maran.
Phenotypic characterization and molecular characterization
All the colonies were identified accordingly to the morphology characteristics as mentioned by EPPO [9]. Colonies were lemon-yellow in colour, circular, raised and convex on King’s B agar medium (Fig 2). From the 28 strains, all of the strains were positive and verified for P. stewartii subsp. stewartii identification (Table 3). PCR amplification of all 28 isolated strains using 16S-23S ITS primer pair, each produced a ~900 bp amplicon (S1 Fig). Phylogenetic analysis based on partial 16S-23S ITS gene (Fig 3) revealed all strains isolated in this study were 100% clustered together with the P. stewartii subsp. stewartii reference strains DC283, S3 and W1. None of the strains clustered with P. agglomerans (Pantoea agglomerans) and P. ananatis (Pantoea anantis) strains.
Bootstrap values after 1000 replicates are expressed as percentages. Pantoea agglomerans and Pantoea ananatis were included as an outgroup. The scale bar indicates the fraction of substitutions per site.
Hypersensitive assay
Hypersensitivity response (HR) induced by all 28 strains at 24 h after inoculation is shown in Fig 4. Necrotic lesions or chlorosis was observed up to 3 days post-infiltration. No changes appeared on the control tobacco plants.
Tobacco infiltration assay of Pantoea stewartii subspecies stewartii strains (a) 12 h after infiltration tobacco leaf inoculated with JEN-14; and (b) 24 h after infiltration tobacco leaf inoculated with JEN-14; (c) Control tobacco plant. The hypersensitive reaction was induced by all Pantoea stewartii subsp. stewartii strains on tobacco plant under the greenhouse condition.
Pathogenicity test
All 28 P. stewartii subsp. stewartii strains produced jackfruit-bronzing disease symptoms within 14 days post-inoculation (Fig 5). No disease symptom appeared on the control. The re-isolation of the inoculated strains were tested further via phenotypic and molecular characterization and showed similar characteristics to positive P. stewartii subsp. stewartii test.
(a) Yellowish discolouration with bronzing specks inoculated with JEN-14 strain; (b) Reddish discolouration inoculated with MAR-D strain; (c) Control jackfruit pulp.
MLSA- single and concatenated gyrB, rpoB, atpD and infB gene sequences
Phylogenetic trees computed based on single gyrB, rpoB, atpD and infB genes (Fig 6), all revealed 99–100% similarities between our 28 bacterial strains with P. stewartii subsp. stewartii reference strains LMG 2713 (USA), LMG 2715 (USA), LMG 2718 (USA) and P. stewartii subsp. indolegenes reference strains PNA 14–12 (USA), LMG 2673 (Hawaii, USA) and LMG 2631 (India) reference strains (Sub-cluster I). Similarly, phylogenetic trees computed based on concatenated (Fig 7) from gyrB, rpoB, atpD and infB genes showed 100% similarities between the 28 bacterial strains with P. stewartii subsp. stewartii reference strains LMG 2713 (USA), LMG 2715 (USA), LMG 2718 (USA) and P. stewartii subsp. indolegenes reference strains PNA 14–12 (USA), LMG 2673 (Hawaii, USA) and LMG 2631 (India) reference strains. However, although the sequence similarity between the 28 bacterial strains with subspecies stewartii and indolegenes were high (100% similarities) (Sub-cluster II), our strains were more closely related to P. stewartii subsp. stewartii (99% similarities) from its close relative P. stewartii subsp. indologenes (Sub-cluster I). All the strains collected from the four collection areas clustered together, showing no variation between the collection area of the bacterial strains.
Maximum-likelihood tree based on the (a) gyrB; (b) rpoB; (c) atpD; and (d) infB genes. Percentage bootstrap values based on 1000 replicates are given at nodes. Tatumella species were included as an outgroup. Bar: 0.05 substitutions per nucleotide position.
Percentage bootstrap values based on 1000 replicates are given at nodes. Tatumella species were included as an outgroup. Bar: 0.05 substitutions per nucleotide position.
Discussion
Systematic identification and characterization were performed for the verification of P. stewartii subsp. stewartii strains from the 4 different jackfruit-bronzing disease outbreak collection area in Peninsular Malaysia. The strains were verified by morphological, phenotypic and molecular characterization test listed by (9). Starting with the isolation and phenotypic characterization, phenotypic tests were performed. Our study showed all 28 strains demonstrated a similar and parallel response to all phenotypic tests and resembled P. stewartii subsp. stewartii characteristics [5,9,17–19]. Motility test is crucial because stated that most bacteria under Pantoea genus are motile due to the presence of peritrichous flagella except for P. stewartii subsp. stewartii [11,23,24]. Indole test is also important in subspecies identification [18,25], as P. stewartii subsp. indolegenes distinguished and identified from P. stewartii subsp. stewartii through the positive result of indole test with Kovac’s reagent. So, the outcome of our indole and motility test results from the 28 strains showed strong positivity towards the identification of P. stewartii subsp. stewartii.
HR on tobacco (Nicotiana tabacum) was performed on P. stewartii subsp. stewartii and shown to be necrotic [14,18,26]. P. stewartii subsp. stewartii contains and carries a specialized protein secretion complex known as type III secretion systems (T3SSs) producing needle-like injectisomes for the delivery of effector proteins into host cells [11,27]. A previous study by Correa et al. (2012) confirmed that P. stewartii subsp. stewartii obtained from maize contains Hrc-Hrp1 as well as Inv-Mxi-Spa type T3SS, which were responsible for the pathogen colonization of susceptible hosts, and transmission from the vector to host plants by the transversal of the thick plant cell wall through the generation of thin pili. This hrp gene (resistant genes) secretion of P. stewartii subsp. stewartii is required to elicit a HR in tobacco leaves [14,28,29]. HR involves rapid reaction, and sometimes it only takes 8–12 h after being inoculated with the bacterial pathogen to become necrotic and eventually die [29]. Our hypersensitivity assay result was similar with previous reports [14,18], T3SSs and hrp genes were believed to promote parasitism and leads to pathogenesis, so other than virulence, tobacco hypersensitivity is also a very convenient way to screen bacterial cultures for pathogenicity [11,30].
Further confirmation was obtained from molecular characterization analysis. Standard differentiation of bacterial species using 16S-23S ITS region was believed to produce better sequence variability than the 16S rDNA gene as 16S rDNA gene cannot be separated easily by using electrophoresis [20]. Primer pair ES16/ESIG2c was tested on P. stewartii (33 strains), other Pantoea spp. (12 strains), related Erwinia spp. (14 strains) and Xanthomonas campestris pv. campestris (1 strain) and showed that only P. stewartii strains and one P. ananas (P. ananatis) strain yielded at the expected amplicons (0.92-kb fragment) [14]. This primer pair also confirmed the jackfruit-bronzing disease from Muadzam Shah plantation of Pahang state was caused by P. stewartii subsp. stewartii [6]. Our study has proven that all our 28 strains yielded at the expected amplicon’s size and sequence similarity of 100% to other P. stewartii subsp. stewartii strains obtained from GenBank database. Phylogenetic analysis constructed using MEGA7 on the ES16/ES1G2c gene sequences showed our strains clustered with the P. stewartii subsp. stewartii reference strains and none of the strains clustered to P. agglomerans and P. ananatis strains.
For the first time, genetic diversity of P. stewartii subsp. stewartii strains causing jackfruit-bronzing disease in Malaysia has been studied via MLSA. MLSA is a genotypic characterization method using the sequences of multiple protein-coding genes of prokaryotes for taxonomic purposes [31]. The stable housekeeping genes that codes for functional proteins make MLSA as a powerful method with a rapid genetic modification in comparison to 16S rDNA/rRNA [31]. Previous genetic diversity studies using 16S rDNA/rRNA reported the clustering and close relationship of P. stewartii subsp. stewartii with P. ananatis and P. agglomerans [32,33], P. stewartii with P. ananatis [34], P. stewartii subsp. stewartii with Erwinia stewartii [35] and P. stewartii subsp. stewartii with P. ananatis, Pantoea deleyi, P. agglomerans, Pantoea vagans, Pantoea eucalypti [36]. Undoubtedly, MLSA overpowers 16S rDNA/rRNA in taxanomic discrepancies in the identification purposes of Pantoea genus [37–39]. Four housekeeping genes were used in this study, as recommended by previous studies on MLSA [40–44]. All phylogenetic analyses in this study were performed with bootstrap replication of 1000, as it supports higher phylogenetic differentiation of Pantoea species [40,45]. The three outgroups were previously known as ‘Japanese species’ of Pantoea, namely Pantoea citrea, Pantoea punctata and Pantoea terrea [40]. However, MLSA indicated that the taxonomic position of these “Japanese species” were clustered together with Tatumella ptyseos, so they were then transferred to the genus Tatumella.
Phylogenetic trees obtained from MLSA based on sequences of gyrB, rpoB, atpD and infB confirmed the allocation of all our strains to the genus Pantoea. Phylogenetic analysis showed that all of the 28 bacterial strains collected from the four collection areas were clustered together and phylogenetically most closely related (bootstrap value of 99–100%) to P. stewartii subsp. stewartii and P. stewartii subsp. indolegenes reference strains. Our result is congruent with the previous studies of MLSA performed on Pantoea species where phylogenetic trees based on housekeeping genes (gyrB, rpoB, atpD and infB) revealed 99–100% similarities between P. stewartii subsp. stewartii and P. stewartii subsp. indolegenes [35,40,45–51].
However, MLSA based on the concatenated genes were able to distinguish that our strains were more closely related to P. stewartii subsp. stewartii (99% similarities) from its close relative P. stewartii subsp. indologenes, although sequence similarity between the subspecies was high (up to 100%). Study of MLSA that performed less than four housekeeping genes were insufficient and more bias, due to low-resolution power or genes impaired [44]. High discriminatory power is gained from MLSA from the concatenated genes than single gene [52]. In other words, the number of genes selection must be considered to achieve more discriminatory power [52].
All bacterial strains collected from the four areas of Malaysia were clustered together with the reference strains, indicating that the strains were genetically similar and share close phylogenetic relatedness with each other. This emphasizes that all our strains were likely originated and derived from a single emergence a long time ago [53]. Based on phylogenetic tree of the concatenated sequences of the four housekeeping genes (Fig 7), no major changes in the clustering pattern appeared since the position of the P. stewartii subsp. indologenes reference strains were still in one cluster (100% similarity) with our bacterial strains and P. stewartii subsp. stewartii reference strains. Hence, in our case, all the 28 P. stewartii subsp. stewartii bacterial strains (single and concatenated) might have a common origin with P. stewartii subsp. stewartii collected from USA (LMG 2713, LMG 2715 and LMG 2718) and P. stewartii subsp. indologenes bacterial strains collected from USA, Hawaii and India (PNA 14–12, LMG 2673 and LMG 2631).
In conclusion, our study verified and confirmed that bacteria causing jackfruit-bronzing in four areas of Malaysia is P. stewartii subsp. stewartii. We highlighted the genetic diversity of P. stewartii subsp. stewartii associated with jackfruit-bronzing in Malaysia, providing new insight on no genetic variation among the collected strains. Phylogenetic analyses indicated that our strains were closely related to P. stewartii subsp. stewartii strains (concatenated genes) and both P. stewartii subsp. stewartii and P. stewartii subsp. indologenes reference strains (single genes). As the improvement in jackfruit industries in Malaysia is vital, once jackfruit-bronzing disease is properly investigated, management options can be deployed to mitigate the disease impact. Controlling plant disease involves proper verification of the causal agent, where in this study, we provide information on the phenotypic tests, hypersensitivity test, pathogenicity test, molecular test, as well as genetic diversity evaluation through MLSA of P. stewartii subsp. stewartii. The output and data of this study will be useful as a proper reference and documentation in quarantine purposes and prevention of the spread of jackfruit-bronzing disease and its causal pathogen, P. stewartii subsp. stewartii.
Supporting information
S1 Table. The expected results of phenotypic tests Pantoea stewartii subspecies stewartii from previous studies.
https://doi.org/10.1371/journal.pone.0234350.s001
(DOCX)
S2 Table. List of amplification primers used in this study.
https://doi.org/10.1371/journal.pone.0234350.s002
(DOCX)
S3 Table. List of sequencing primers used in this study.
https://doi.org/10.1371/journal.pone.0234350.s003
(DOCX)
S4 Table. Origin and characterization of Pantoea stewartii subspecies stewartii strains from Malaysia and reference strains for 16S-23S Internally Transcribed Spacer (ITS) Region in this study.
https://doi.org/10.1371/journal.pone.0234350.s004
(DOCX)
S5 Table. Origin and characterization of Pantoea stewartii subspecies stewartii strains from Malaysia and reference strains for MLSA in this study.
https://doi.org/10.1371/journal.pone.0234350.s005
(DOCX)
S1 Fig. Agarose gels showing PCR product from 16S-23S Internally Transcribed Spacer (ITS) region.
Size of DNA ladder (M) used was 100bp (GeneDireX, Inc.) The amplification fragments were ~900 bp. (a) Strains from Jenderam; (b) Strains from Maran; (c) Strains from Ipoh; (d) Strains from Muadzam Shah; and -NC represent the Negative control.
https://doi.org/10.1371/journal.pone.0234350.s006
(TIF)
References
- 1. Sundarraj AA, Ranganathan thottiam V. Utilization of Jackfruit Wastes for the Production of Food Additives View project Preparation of Laminated Baked Product using Oats View project. Vegetos. 2018;31: 67–73.
- 2.
PEMANDU. Economic Transformation Programme: Annual Report 2012. Malaysia; 2012. Available: https://www.pmo.gov.my/dokumenattached/Eng_ETP2012_Full.pdf
- 3.
KADA. Varieties Registered For National Crop List. Kemubu Agricultural Development Authority, Malaysia; 2018. Available: http://pvpbkkt.doa.gov.my/NationalList/Search.php
- 4.
Halim NA. Policy Intervention for the Development of the Pineapple Industry in Malaysia. In: FFTC Agricultural Policy Article [Internet]. Malaysia; 2016 [cited 6 Aug 2019]. Available: http://ap.fftc.agnet.org/ap_db.php?id=716
- 5. Gapasin RM, Garcia RP, Christine T, Cruz CS De, Borines LM. Fruit Bronzing: a New Disease Affecting Jackfruit Caused by Pantoea stewartii (Smith) Mergaert et al. Ann Trop Res. 2014;36: 17–31.
- 6. Zulperi D, Manaf N, Ismail SI, Karam DS, Yusof MT. First Report of Pantoea stewartii subsp. stewartii Causing Fruit Bronzing of Jackfruit (Artocarpus heterophyllus), a New Emerging Disease in Peninsular Malaysia. Plant Dis. 2017;101: 831–831.
- 7. Hernández-Morales A, Pérez-Casillas JM, Soria-Guerra RE, VelázquezFernández JB, Arvizu-Gómez JL. First report of Pantoea stewartii subsp. stewartii causing jackfruit bronzing disease in Mexico. J Plant Pathol. 2017;99: 807.
- 8. ITFN. Rust-like symptoms and rot on jackfruit: A combination of disease and abiotic factors?. ITFN Tropical Fruit Net Newsletter. 2012: 16–17.
- 9. EPPO. PM 7/60 (2) Pantoea stewartii subsp. stewartii. EPPO Bull. 2016;46: 226–236.
- 10. Jeger M, Bragard C, Candresse T, Chatzivassiliou E, Dehnen‐Schmutz K, Gilioli G, et al. Pest categorisation of Pantoea stewartii subsp. stewartii. EFSA J. 2018;16.
- 11. Roper MC. Pantoea stewartii subsp. stewartii: Lessons learned from a xylem-dwelling pathogen of sweet corn. Mol Plant Pathol. 2011;12: 628–637. pmid:21726365
- 12. EPPO. Pantoea stewartii subsp. stewartii. PM 7/60(1) OEPP/EPPO Bull. 2006;36: 111–115. Available: www.eppo.org.
- 13. Goszczynska T, Botha WJ, Venter SN, Coutinho TA. Isolation and Identification of the Causal Agent of Brown Stalk Rot, A New Disease of Maize in South Africa. Plant Dis. 2007;91: 711–718. pmid:30780480
- 14. Coplin DL, Majerczak DR, Zhang Y, Kim W-S, Jock S, Geider K. Identification of Pantoea stewartii subsp. stewartii by PCR and strain differentiation by PFGE. 2002. pmid:30818612
- 15. Kini K, Agnimonhan R, Dossa R, Silué D, Koebnik R. A diagnostic multiplex PCR scheme for identification of plant-associated bacteria of the genus Pantoea. 2018 [cited 8 Jan 2019].
- 16.
Vinuesa P. Molecular Phylogeny of Microorganisms. Papke T, editor. Norfolk, UK: Caister Academic Press; 2010.
- 17. Rahma H, Sinaga MS, Surahman M. First Report of Stewart’s Wilt of Maize Caused by Pantoea stewartii subsp. stewartii In Bogor District, Indonesia. J Int Soc Southeast Asian Agric Sci. 2014;20: 131–141.
- 18. Gehring I, Wensing A, Gernold M, Wiedemann W, Coplin DL, Geider K. Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds. J Appl Microbiol. 2014;116: 1553–1562. pmid:24905218
- 19. Orio AGA, Brücher E, Plazas MC, Sayago P, Guerra F, De Rossi R, et al. First Report of Stewart’s wilt of maize in Argentina caused by Pantoea stewartii. Plant Dis. 2012;96: 1819–1819. pmid:30727295
- 20. Suryani L, Aini LQ, Sugiharto AN, Abadi AL. Characterization of bacterial pathogen causing wilt and leaf blight on corn (Zea mays) by physiological, biochemical and molecular methods. Agrivita Vol. 2012;34: 286–295.
- 21. Hall AT. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41: 95–98.
- 22. Kumar S, Stecher G, Tamura K, Dudley J. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33: 1870–1874. pmid:27004904
- 23.
Goszczynska T, Serfontein JJ, Serfontein S. Introduction to practical phytobacteriology: a manual for phytobacteriology. ARC—Plant Protection Research Institute; 1999.
- 24.
Gutierrez CMH. Pantoea stewartii subsp. stewartii requires motility for plant infection. PhD Thesis. University of Connecticut. 2008.
- 25. Nechwatal J, Friedrich-Zorn M, Theil S, Gebauer P, Wensing A. Validation of a specific PCR screening test for Pantoea stewartii subsp. stewartii in maize (Zea mays) samples. EPPO Bull. 2018;48: 78–85.
- 26. Ham JH, Majerczak D, Ewert S, Sreerekha M-V, Mackey D, Coplin D. WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants. Mol Plant Pathol. 2008;9: 633–643. pmid:19018993
- 27. Correa VR, Majerczak DR, Ammar E-D, Merighi M, Pratt RC, Hogenhout SA, et al. The Bacterium Pantoea stewartii Uses Two Different Type III Secretion Systems To Colonize Its Plant Host and Insect Vector Downloaded from. 2012 [cited 26 Mar 2019]. pmid:22773631
- 28. Frederick RD, Ahmad M, Majerczak DR, Arroyo-Rodríguez AS, Manulis S, Coplin DL. Genetic Organization of the Pantoea stewartii subsp. stewartii hrp Gene Cluster and Sequence Analysis of the hrpA, hrpC, hrpN, and wtsE Operons. Mol Plant-Microbe Interact. 2001;14: 1213–1222. pmid:11605961
- 29.
Janse JD. Phytobacteriology: principles and practice. Wallingford: CAB International; 2005. Available: http://agris.fao.org/agris-search/search.do?recordID = XF2015036287
- 30. Mulaw T, Wamishe Y, Jia Y. Characterization and in Plant Detection of Bacteria that Cause Bacterial Panicle Blight of Rice. Am J Plant Sci. 2018;9: 667–684.
- 31. Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coenye T, Feil EJ, et al. Re-evaluating prokaryotic species. Nat Rev Microbiol. 2005;3: 733–739. pmid:16138101
- 32. Naum M, Brown EW, Mason-Gamer RJ. Is 16S rDNA a reliable phylogenetic marker to characterize relationships below the family level in the enterobacteriaceae? J Mol Evol. 2008;66: 630–642. pmid:18504519
- 33. Young JM, Park DC. Relationships of plant pathogenic enterobacteria based on partial atpD, carA and recA as individual and concatenated nucleotide and peptide sequences. Syst Appl Microbiol. 2007;30: 343–354. pmid:17451899
- 34. Wang L, Wang J, Jing C. Comparative Genomic Analysis Reveals Organization, Function and Evolution of ars Genes in Pantoea spp. Front Microbiol. 2017;8. pmid:28377759
- 35. Yazdani R, Safaie N, Shams-Bakhsh M. Association of Pantoea ananatis and Pantoea agglomerans with leaf spot disease on ornamental plants of Araceae Family. Eur J Plant Pathol. 2018;150: 167–178.
- 36. Castagno LN, Estrella MJ, Sannazzaro AI, Grassano AE, Ruiz OA. Phosphate-solubilization mechanism and in vitro plant growth promotion activity mediated by Pantoea eucalypti isolated from Lotus tenuis rhizosphere in the Salado River Basin (Argentina). J Appl Microbiol. 2011;110: 1151–1165. pmid:21299771
- 37. Palmer M, Steenkamp ET, Coetzee MPA, Chan WY, van Zyl E, De Maayer P, et al. Phylogenomic resolution of the bacterial genus Pantoea and its relationship with Erwinia and Tatumella. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2017;110: 1287–1309. pmid:28255640
- 38. Gutiérrez-Barranquero JA, Cazorla FM, Torés JA, de Vicente A. Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees. Phytopathology. 2019;109: 17–26. pmid:30102576
- 39. Delétoile A, Decré D, Courant S, Passet V, Audo J, Grimont P, et al. Phylogeny and Identification of Pantoea species and Typing of Pantoea agglomerans Strains by Multilocus Gene Sequencing. J Clin Microbiol. 2009;47: 300–310. pmid:19052179
- 40. Brady C, Cleenwerck I, Venter S, Vancanneyt M, Swings J, Coutinho T. Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA). Syst Appl Microbiol. 2008;31: 447–460. pmid:19008066
- 41. Guo Y, Zheng W, Rong X, Huang Y. A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics. Int J Syst Evol Microbiol. 2008;58: 149–159. pmid:18175701
- 42. Martens M, Dawyndt P, Coopman R, Gillis M, De Vos P, Willems A. Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int J Syst Evol Microbiol. 2008;58: 200–214. pmid:18175710
- 43. Kuhnert P, Korczak BM, Stephan R, Joosten H. Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA). Int J Food Microbiol. 2009;136: 152–158. pmid:19321218
- 44. Glaeser SP, Kämpfer P. Multilocus sequence analysis (MLSA) in prokaryotic taxonomy. Syst Appl Microbiol. 2015;38: 237–245. pmid:25959541
- 45. Popp A, Cleenwerck I, Iversen C, De Vos P, Stephan R, Roger C, et al. Pantoea gaviniae sp. nov. and Pantoea calida sp. nov., isolated from infant formula and an infant formula production environment. Int J Syst Evol Microbiol. 2010;60: 2786–2792. pmid:20061487
- 46. Brady C, Cleenwerck I, Venter SN, Engelbeen K, De Vos P, Coutinho TA. Emended description of the genus Pantoea, description of four species from human clinical samples, Pantoea septica sp. nov., Pantoea eucrina sp. nov., Pantoea brenneri sp. nov. and Pantoea conspicua sp. nov. Int J Syst Evol Microbiol. 2010;60: 2430–2440. pmid:19946052
- 47. Kvitko BH, Gitaitis R, Dutta B. Isolation and Characterization of novel Pantoea stewartii subsp. indologenes strains exhibiting center rot in onion. Plant Dis. 2017;102: 727–733.
- 48. Xu R, Chen Q, Robleh Djama Z, Tambong JT. Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii. Lett Appl Microbiol. 2010;50: 216–222. pmid:20002575
- 49. Chen C, Xin K, Liu H, Cheng J, Shen X, Wang Y, et al. Pantoea alhagi, a novel endophytic bacterium with ability to improve growth and drought tolerance in wheat. Sci Rep. 2017;7. pmid:28128318
- 50. Duron O, Noël V. A wide diversity of Pantoea lineages are engaged in mutualistic symbiosis and cospeciation processes with stinkbugs. Environ Microbiol Rep. 2016;8: 715–727. pmid:27362408
- 51. Zhang Y, Qiu S. Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2015;108: 1037–1046. pmid:26296376
- 52. De Vos P. Multilocus Sequence Determination and Analysis. Methods Microbiol. 2011;38: 385–407.
- 53. Moretti C, Vinatzer BA, Onofri A, Valentini F, Buonaurio R. Genetic and phenotypic diversity of Mediterranean populations of the olive knot pathogen, Pseudomonas savastanoi pv. savastanoi. Plant Pathol. 2017;66: 595–605.