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Genomics-Informed Multiplex PCR Scheme for Rapid Identification of Rice-Associated Bacteria of the Genus Pantoea

    Affiliations
    Authors and Affiliations
    • Kossi Kini1 2
    • Raoul Agnimonhan1
    • Rachelle Dossa1
    • Drissa Silué1 3
    • Ralf Koebnik2 4 †
    1. 1Africa Rice Center (AfricaRice), Cotonou, Benin
    2. 2IRD, Cirad, University Montpellier, IPME, Montpellier, France
    3. 3Africa Rice Center (AfricaRice), M’be Research Center, Bouake, Côte d’Ivoire
    4. 4Plant Health Institute of Montpellier (PHIM), Univ Montpellier, Cirad, INRAe, Institut Agro, IRD, Montpellier, France
    Published Online:14 Oct 2021https://doi.org/10.1094/PDIS-07-20-1474-RE

    Abstract

    The genus Pantoea forms a complex of more than 25 species, among which several cause diseases of various crop plants, including rice. Notably, strains of Pantoea ananatis and P. stewartii have been repeatedly reported to cause bacterial leaf blight of rice, whereas other authors have observed that P. agglomerans can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools. Using 32 whole-genome sequences of the three major plant-pathogenic Pantoea spp., a set of PCR primers that detect each of the three species P. agglomerans, P. ananatis, and P. stewartii was designed. A multiplex PCR scheme which can distinguish these three species and also detects members of other Pantoea spp. was further developed. Upon validation on a set of reference strains, 607 suspected Pantoea strains that were isolated from rice leaves or seed originating from 11 African countries were screened. In total, 41 P. agglomerans strains from 8 countries, 79 P. ananatis strains from 9 countries, 269 P. stewartii strains from 9 countries, and 218 unresolved Pantoea strains from 10 countries were identified. The PCR protocol allowed detection of Pantoea bacteria grown in vitro, in planta, and in rice seed. The detection threshold was estimated as total genomic DNA at 0.5 ng/µl and heated cells at 1 × 104 CFU/ml. This new molecular diagnostic tool will help to accurately diagnose major plant-pathogenic species of Pantoea. Due to its robustness, specificity, sensitivity, and cost efficiency, it will be very useful for plant protection services and for the epidemiological surveillance of these important crop-threatening bacteria.

    The genus Pantoea was first described in 1989 and was recently taxonomically classified as a member of the Erwiniaceae family (Adeolu et al. 2016; Gavini et al. 1989). More than 25 species of this genus have been described and reported worldwide (Brady et al. 2008; Mergaert et al. 1993; Tambong 2019). Etymologically, the genus name Pantoea is derived from the Greek word ‘Pantoios’, which means “of all sorts or sources” and reflects the diverse geographical and ecological sources from which the bacteria have been isolated. Several species of the genus are qualified as versatile and ubiquitous bacteria because they have been isolated from many different ecological niches and hosts (Brady et al. 2008, 2010a). Remarkably, some species have the ability to colonize and interact with members of both the plant and the animal kingdoms (De Maayer et al. 2014).

    A few species of Pantoea are responsible for several diseases of plants, including important crops such as rice, maize, sorghum, onion, and cotton (Brady et al. 2008; Coutinho and Venter 2009; Dutkiewicz et al. 2016; Walterson and Stavrinides 2015; Weller-Stuart et al. 2017). Three species of Pantoea—namely, Pantoea agglomerans, P. ananatis, and P. stewartii—have been repeatedly isolated from symptomatic rice samples throughout the world (e.g., in Australia, Korea, China, India, Russia, Venezuela, Benin, and Togo) (Cother et al. 2004; Egorova et al. 2015; González et al. 2015; Kini et al. 2017a, b; Lee et al. 2010; Mondal et al. 2011; Yan et al. 2010).

    Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae, is an important disease of rice and affects rice cultivation in most regions of the world where rice is grown (Triplett et al. 2014). The bacterium has been associated with this disease for more than 100 years (Niño-Liu et al. 2006). Surveys conducted from 2010 to 2016 by AfricaRice and its partners aimed at estimating the extent and importance of the disease and the phytosanitary status of rice fields in West Africa. Although leaves showing BLB-like symptoms were frequent, isolation or molecular detection of xanthomonads using a common multiplex PCR diagnostic tool (Lang et al. 2010) often failed. Instead, other bacteria forming yellow colonies were observed that belong to the species P. ananatis or P. stewartii, as documented for samples from Benin and Togo (Kini et al. 2017a, b). Moreover, other cases of BLB and grain discoloration caused by Sphingomonas spp. and other undescribed species have been detected in several sub-Saharan African countries (Kini et al. 2017c). This situation of emerging bacterial pathogens that constitute a threat to rice production in Africa calls for a robust, specific, sensitive, and cost-efficient diagnostic tool for accurate pathogen detection. However, none of the physiological, biochemical, or molecular diagnostic tools for Pantoea, including several simplex and multiplex PCR tools, allowed the accurate and simultaneous detection of three major plant-pathogenic Pantoea spp. (P. agglomerans, P. ananatis, and P. stewartii) (Asselin et al. 2016; Brady et al. 2007; Coplin et al. 2002; Feng et al. 2015; Pataky et al. 2004; Rezzonico et al. 2010; Thapa et al. 2012; Uematsu et al. 2015; Xu et al. 2010; Zhao et al. 2014).

    To overcome this situation, a molecular method was devised for detecting these three important plant-pathogenic Pantoea spp., as well as other members of the genus, in a single reaction. A universal multiplex PCR tool was developed and first tested in silico on available genome sequences and on a set of reference strains from the United States, Brazil, Spain, and Japan. Afterward, 607 suspected Pantoea strains from 11 Africans countries were evaluated with this new diagnostic tool. P. agglomerans was detected in rice leaves from several African countries for the first time. Finally, the specificity and sensitivity of the multiplex PCR was monitored by analyzing serial dilutions of genomic DNA, serial dilutions of bacterial cell suspensions, and solutions of ground leaves and seed that had been artificially or naturally infected. This new diagnostic tool will prove useful for phytosanitary services in routine diagnostics of Pantoea spp. in any type of sample (e.g., leaves, seed, soil, or water).

    Materials and Methods

    Bioinformatics prediction of specific PCR primers.

    Pantoea genome sequences were retrieved from NCBI GenBank (Table 1). Sequences for housekeeping genes were identified by TBLASTN. Sequences were then aligned with MUSCLE at EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/muscle/). Diagnostic primers that can differentiate the three species P. agglomerans, P. ananatis, and P. stewartii, and one primer pair that would amplify DNA from the whole Pantoea genus were designed manually. Primer melting temperature (Tm) was calculated with the Multiple Primer Analyzer Tm calculator tool (Thermo Fisher Scientific 2021).

    Table 1. List of Pantoea genome sequences used for primers design

    Sample preparation.

    Different types of samples, including total genomic DNA, bacterial cells, and symptomatic rice leaves, as well as discolored or apparently healthy rice seed, were analyzed. Bacterial colonies were grown for 24 to 48 h on peptone-sucrose-agar (PSA) plates containing 10 g of peptone, 10 g of sucrose, 16 g of agar, and 1 g of glutamic acid per liter. Total genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega, Charbonnières-les-Bains, France) according to the manufacturer’s instructions. DNA quality and quantity were evaluated by agarose gel electrophoresis and spectrophotometry (Nanodrop Technologies, Wilmington, DE, U.S.A.).

    When using bacterial cells as PCR template, bacteria were freshly grown on PSA plates for 24 h at 28°C. Cells from 1 cm2 of the bacterial lawn were then resuspended in 1 ml of sterilized distilled water. For template preparation, 10 μl of the bacterial suspension were diluted in 90 μl of sterilized distilled water and incubated at 95°C for 15 min.

    To evaluate the PCR scheme on live plant material, leaves and seed were artificially inoculated with strains of the three Pantoea spp. Rice leaves of the cultivar Azucena were inoculated as described previously (Kini et al. 2017a, b). To produce contaminated seed, early maturity panicles of the Azucena rice cultivar were spray inoculated with a 5%-gelatinized bacterial solution (106 CFU/ml). Distillated and gelatinized (5%) sterile water served as the negative control. Three weeks postinoculation, approximately 40% of the grains in the panicles exhibited discolorations. Panicles inoculated with sterile distilled water showed no symptoms. Plant material was ground and macerated before use as PCR template. For this purpose, five grains or 0.5 g of symptomatic rice leaf material were surface sterilized with a solution of hypochlorite (10%) and ethanol (70%), rinsed with sterile distilled water, and ground in 100 ml of sterile distilled water. After centrifugation (3,000 rpm for 5 min), 10 μl of the supernatant were diluted in 90 μl of sterile distilled water and incubated at 95°C for 15 min before setup of the PCR.

    Optimization of the multiplex PCR.

    To develop a multiplex PCR scheme, individual primer pairs were first tested against the different samples mentioned above, using annealing temperatures close to the predicted Tm (Tm ± 5°C) and with a progressive number of PCR cycles (25 to 35). Primer pairs were then mixed from duplex to quintuplex and PCR conditions were evaluated, testing annealing temperatures close to the optimal Tm of the individual primer pairs (Tm ± 3°C) and various numbers of PCR cycles. At the end, three promising combinations of annealing temperatures and numbers of PCR cycles were reevaluated in simplex PCR with the samples mentioned above. The best combination with high specificity and without background amplification was selected as the new diagnostic tool.

    Both simplex and multiplex PCR were performed in 25-µl buffered solutions containing 50 µM each of the four dNTPs, 0.16 µM each of the oligonucleotides (Table 2), 0.5 U of Takara ExTaq (Takara Bio Europe SAS, Saint-Germain-en-Laye, France), and 2 µl of template sample (genomic DNA or heat-treated cell suspension). PCR protocols involved a 3-min initial denaturation step at 94°C; 30 cycles consisting of denaturation (30 s at 94°C), annealing (30 s at 58°C), and extension (2 min at 72°C) steps; and a final extension period of 10 min at 72°C. PCR products were electrophoretically separated on 1.5% (wt/vol) agarose gels and visualized under UV light upon staining with ethidium bromide.

    Table 2. List of PCR primers of the Pantoea-specific multiplex PCR scheme

    Evaluation of the sensitivity of the multiplex PCR scheme using genomic DNA and heat-treated cells.

    Simplex and multiplex PCR were used to evaluate the sensitivity of all of the species-specific primer pairs individually or in combination with the genus-specific and the 16S ribosomal RNA (rRNA) primer pairs. Serial dilutions of total genomic DNA and heated bacterial cells were used for this evaluation (Aneja 2005). Three Pantoea strains—P. ananatis strain ARC60, P. stewartii strain ARC229, and P. agglomerans strain CFBP 3615—were used, and distilled sterilized water served as the negative control.

    Evaluation of the multiplex PCR scheme on a large collection of African Pantoea strains.

    Bacterial strains used in this study are listed in Supplementary Table S1. In total, 615 Pantoea strains from 11 Africans countries (Benin, Burkina Faso, Burundi, Ghana, Ivory Coast, Mali, Niger, Nigeria, Senegal, Tanzania, and Togo) and 7 reference strains from the United States, Brazil, Spain, and Japan were tested with the new diagnostic tool. The African strains were isolated from rice leaves with BLB symptoms and from discolored or apparently healthy rice seed. The samples had been collected from 2008 to 2016 in the main rice-growing areas of the countries. The strains were purified as single colonies, individually grown, and preserved as pure cultures following routine methods (Poulin et al. 2014).

    Results

    Development of a diagnostic PCR scheme for plant-associated Pantoea spp.

    The goal of this study was to design diagnostic PCR primers that would target conserved housekeeping genes. The rationale behind this was that these genes should be present in all strains, including genetic lineages that have not yet been discovered and would not be present in any strain collection. At the same time, we knew from previous work that sequences of housekeeping genes are divergent enough to doubtlessly distinguish and identify Pantoea strains at the species level.

    A diagnostic Pantoea multiplex PCR method was developed in two steps. First, a complete inventory of publicly available Pantoea genome sequences was compiled, consisting of 9 P. agglomerans, 19 P. ananatis, and 4 P. stewartii sequences, totaling 32 whole-genome sequences (Table 1). Complete coding sequences of four housekeeping genes that have previously been used for multilocus sequence analyses (MLSA) of Pantoea spp. (Brady et al. 2008)—atpD, gyrB, infB, and rpoB—were then extracted and aligned. Sequence regions that were conserved in all strains of one species but were significantly different in the other two species were identified manually and chosen to design PCR primers (Table 2). An important criterium was that most mismatches should be present close to the 3′ end of the primers (Supplementary Figures S1 to S4).

    To enable multiplexing, we made sure that the amplicon sizes would be between 400 and 750 bp and vary in size enough to be easily distinguishable from each other upon agarose gel electrophoresis (Fig. 1). As a positive control for the PCR, one primer pair was included that would amplify DNA from all bacteria belonging to the Pantoea genus, resulting in a smaller amplicon of less than 400 bp. Finally, as a second control, a primer pair was included that targets the 16S rRNA gene and leads to an amplicon that is larger than the four Pantoea-specific amplicons. Upon testing several combinations, the finally selected species-specific primer pairs had at least seven mismatches in the other two species, thus avoiding any cross reactivity (Supplementary Figures S1 to S4).

    Fig. 1.

    Fig. 1. Schematic representation of the multiplex PCR scheme. Sizes of the five expected PCR amplicons are indicated in the middle and their expected migration in a 1.5% Tris-borate-EDTA agarose gel is shown on the left side. Diagnostic band patterns for the three plant-associated Pantoea spp. are shown on the right side. PANAN = Pantoea ananatis, PANST = P. stewartii, and PANAG = P. agglomerans.

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    In the next step, all primer pairs (Table 2) were evaluated, first by simplex PCR and then by multiplex PCR, with increasing number of primer pairs, as explained in Materials and Methods. Three Pantoea reference strains were used to develop the PCR scheme using genomic DNA and heat-inactivated bacteria: P. agglomerans strain CFBP 3615, P. ananatis strain ARC60, and P. stewartii strain ARC229 (Fig. 2). Agarose gel electrophoresis demonstrated that the multiplex PCR was able to detect and distinguish all three Pantoea spp. Notably, the multiplex PCR scheme was also able to detect two or three Pantoea spp. when the corresponding species were present in the same template DNA, as demonstrated by PCR assays containing equal amounts of DNA of the different species (Fig. 2).

    Fig. 2.

    Fig. 2. Detection of three Pantoea spp. by multiplex PCR, using heated cell suspensions or genomic DNA as template. Three reference strains were used as representatives for the three Pantoea spp.: Pantoea ananatis strain ARC60 (PANAN), P. stewartii strain ARC229 (PANST), and P. agglomerans strain CFBP 3615 (PANAG). Sizes of the five expected PCR amplicons are indicated on the right side. Lanes 1 and 5, pool of heated cells of the three Pantoea spp.; lane 2, P. ananatis; lane 3, P. stewartii; and lane 4, pool of genomic DNA from P. ananatis and P. agglomerans.

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    Validation of the diagnostic PCR scheme on artificially inoculated plant samples.

    To simplify the analyses and to avoid the isolation of bacteria from plant samples, the PCR scheme was also evaluated on infected leaf material and contaminated seed, thus reducing the costs per sample. For this purpose, rice leaves of a susceptible cultivar were artificially inoculated with a bacterial suspension and symptomatic leaf samples were collected once symptoms were visible. Similarly, early-maturity panicles of a susceptible rice cultivar were spray inoculated with a bacterial suspension and grains were collected 3 weeks postinoculation. The multiplex PCR was able to doubtlessly detect all three Pantoea spp. in both types of plant samples (Fig. 3), as demonstrated for the strains CFBP 3615 (P. agglomerans), ARC60 (P. ananatis), and ARC229 (P. stewartii). Thus, a robust PCR-based method was established that was able to amplify DNA from total genomic DNA, from bacterial cells, from symptomatic rice leaves, and from infected rice seed.

    Fig. 3.

    Fig. 3. Detection of three Pantoea spp. in artificially infected rice leaves and in contaminated seed. The following Pantoea strains were used: Pantoea ananatis strain ARC60 (PANAN), P. stewartii strain ARC229 (PANST), and P. agglomerans strain CFBP 3615 (PANAG). Lane 1, P. ananatis (leaf sample); lane 2, P. ananatis (seed); lane 3, P. stewartii (leaf); lane 4, P. stewartii (seed); lane 5, P. agglomerans (leaf); lane 6, P. agglomerans (seed); lane 7, a yellow bacterial colony isolated from rice seed, probably an undefined Pantoea spp.; and lane 8, water.

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    Evaluation of the sensitivity of the multiplex PCR scheme using genomic DNA and heated cell suspensions.

    The evaluation by simplex and multiplex PCR showed that all of the species-specific primers were very sensitive individually or in combination with the genus-specific and the 16S rRNA universal primers (Fig. 4). Under our experimental conditions, the most sensitive primer pair in simplex PCR was the one targeting P. stewartii, with a detection threshold of 5 pg/µl, followed by the P. agglomerans-specific primer pair (detection threshold of 50 pg/µl) and the P. ananatis-specific primer pair (detection threshold of 0.5 ng/µl). A similar trend was observed in the multiplex PCR on genomic DNA, with the same detection limit as in simplex PCR for P. stewartii and P. ananatis and 10-fold less sensitivity for P. agglomerans.

    Fig. 4.

    Fig. 4. Sensitivity of PCR amplification in simplex and multiplex PCR. Serial dilutions of total genomic DNA and heated bacterial cells were evaluated. I, Simplex PCR with bacterial cells; II, multiplex PCR with bacterial cells; III, simplex PCR with genomic DNA; and IV, multiplex PCR with genomic DNA. Three Pantoea strains were used: Pantoea ananatis strain ARC60 (row A), P. stewartii strain ARC229 (row B), and P. agglomerans strain CFBP 3615 (row C). Simplex PCR were performed with the corresponding, species-specific primer pairs: PANAN_gyrB for P. ananatis, PANST_rpoB for P. stewartii, and PANAG_infB for P. agglomerans. The multiplex PCR included all five primer pairs. The following amounts of bacteria or genomic DNA were used as templates for the PCR, corresponding to 10-fold serial dilutions: lanes 1 to 12, 106 CFU/ml, 105 CFU/ml, 104 CFU/ml, 103 CFU/ml, 102 CFU/ml, 101 CFU/ml, 10° CFU/ml, 10−1 CFU/ml, 10−2 CFU/ml, 10−3 CFU/ml, 10−4 CFU/ml, and water; lanes 13 to 24, 50/µl ng, 5 ng/µl, 0.5 ng/µl, 50 pg/µl, 5 pg/µl, 0.5 pg/µl, 50 fg/µl, 5 fg/µl, 0.5 fg/µl, 50 ag/µl, 5 ag/µl, and water; and lane M, molecular size marker (1-kb DNA ladder, from 10 kb to 1,000 bp, 750 bp, 500 bp, and 250/253 bp [double band]) (Promega). Expected amplicon sizes are indicated on the right side.

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    When heated bacterial cell suspensions were used as template, the P. ananatis-specific primer pair was the most sensitive, allowing detection of 103 CFU/ml, while the other two primer pairs were able to detect 104 CFU/ml. However, when all five primers pairs were used in multiplex, the sensitivity was very similar for all three species, with a detection limit of approximately 104 CFU/ml.

    Evaluation of the multiplex PCR scheme on a large collection of African Pantoea strains.

    Because recent surveys had indicated that Pantoea spp. could be responsible for many unsolved infections of rice fields in sub-Saharan Africa (Kini et al. 2017a, b), we screened a large collection of these isolates. We first reevaluated a few African strains that had been identified as P. ananatis (ARC22, ARC60, and ARC651) and P. stewartii (ARC229, ARC570, and ARC646), using species-specific and the genus-specific PCR primers (Kini et al. 2017a, b). The multiplex PCR scheme confirmed their previous taxonomic classification. Next, we screened a large collection of African bacterial isolates from rice samples (>1,000 strains), among which 609 strains were found to belong to the genus Pantoea (Supplementary Table S1). Specifically, this work diagnosed 41 P. agglomerans strains from 8 countries (Benin, Ghana, Mali, Niger, Nigeria, Senegal, Tanzania, and Togo), 79 P. ananatis strains from 9 countries (Benin, Burkina Faso, Burundi, Mali, Niger, Nigeria, Senegal, Tanzania, and Togo), 269 P. stewartii strains from 9 countries (Benin, Burkina Faso, Ivory Coast, Mali, Niger, Nigeria, Senegal, Tanzania, and Togo), and 218 Pantoea spp. strains from 10 countries (Benin, Burundi, Ghana, Ivory Coast, Mali, Niger, Nigeria, Senegal, Tanzania, and Togo) (Supplementary Table S1). This result provides the first insights into the presence and prevalence of three important Pantoea spp. in these 11 African countries.

    Discussion

    Given the fact that more than 25 species of Pantoea are currently known, among which several species can infect plants, efficient diagnostic tools are highly demanded by plant pathologists and extension workers. Several plant diseases were attributed to only 3 of the >25 species of Pantoea (namely, P. agglomerans, P. ananatis, and P. stewartii) which, therefore, can be considered as the major Pantoea spp. infecting plants. For their diagnosis, several PCR methods are available and have been used but some of them produced amplicons with other species as well (Coplin et al. 2002; Figueiredo and Paccola-Meirelles 2012; Ma et al. 2016), whereas others are not reproducible or are inaccessible in a typical sub-Saharan laboratory due to the lack of specific equipment requirements or high costs of some reagents (Coplin et al. 2002; Uematsu et al. 2015; Xu et al. 2010). Notably, most assays target only one Pantoea sp. or subspecies. For instance, being of major concern, P. stewartii subsp. stewartii causing Stewart’s bacterial wilt can be detected by several methods but none of them can, at the same time, identify other bacteria of the genus Pantoea (Coplin et al. 2002; Gehring et al. 2014; Tambong 2015; Thapa et al. 2012; Xu et al. 2010). To the best of our knowledge, no robust diagnostic scheme exists that can simultaneously and specifically detect these three major Pantoea spp. that infect plants.

    Guided by whole-genome sequences, we developed a robust multiplex PCR scheme that can specifically detect these three important Pantoea spp. Different strategies can be followed when developing such a multiplex scheme. One possibility is to automate the procedure by identifying genomic regions that are shared among a set of strains (e.g., the target species) and which are absent in another set of strains (nontarget species). For instance, such an approach was used for the development of an X. oryzae-specific multiplex PCR scheme that can differentiate the two X. oryzae pathovars oryzae and oryzicola (Lang et al. 2010). The problem with this approach is that it often identifies nonessential, perhaps hypothetical genes as targets for the primer design. Although present in the training set, it is hard to predict whether these nonessential genes are present and conserved in other, hitherto uncharacterized strains, especially when they originate from other geographical zones or belong to more distant genetic lineages.

    Here, we targeted housekeeping genes, which are conserved throughout the genus, and relied on lineage (species)-specific sequence polymorphisms. This approach is considered as very robust but it cannot be ruled out that recombination events among strains from different species could undermine the universality of these primer pairs. However, we did not find any evidence for such events in any of the sequenced Pantoea strains that were analyzed, including isolates from the environment or from human or plant samples. Nevertheless, because this study was focused on isolates from African rice leaves and seed and only included a few reference strains from other continents (Supplementary Table S1), it might be of interest to evaluate the new multiplex PCR tool on Pantoea strains isolated from other organisms (others plants, insects, other animals, and humans) and from environmental samples.

    In addition to the three targeted Pantoea spp., we also checked whether the primers would lead to DNA amplification with other Pantoea spp. or its sister clades, species of the genera Mixta and Tatumella (Brady et al. 2010b; Palmer et al. 2018). For this analysis, we used genome sequences of type strains (Supplementary Table S2). The P. ananatis-specific primer pair (gyrB) was found to have at least 6 mismatches for nontarget Pantoea spp., ≥10 for Mixta, and ≥12 for Tatumella. Similarly, the P. stewartii-specific primer pair (rpoB) was found to have at least 6 mismatches for nontarget Pantoea spp., ≥9 for Mixta, and ≥8 for Tatumella. In contrast, the P. agglomerans-specific primer pair (infB) was found to match as well with the P. vagans sequence and to have only one mismatch for P. anthophila, P. conspicua, P. deleyi, and P. eucalypti; two mismatches for P. dispersa, P. septica, and P. wallisii; and three mismatches for P. cypripedii. Consequently, depending on the position of the mismatches, this primer pair may also amplify DNA beyond P. agglomerans but, importantly, it won’t do so with P. ananatis or P. stewartii. Our comparisons show as well that the genus-specific primer pair (atpD) should amplify DNA from all analyzed Pantoea spp. but it may do so as well with species of Mixta, which is the closest neighbor of Pantoea, and also with some species of Tatumella (Supplementary Table S2). Given that P. agglomerans, P. ananatis, and P. stewartii are among the most important plant-pathogenic species, these extensions of taxonomic specificity do not hamper the utility of this diagnostic tool. However, if necessary, one can easily sequence the atpD or infB amplicons to differentiate among these species and obtain a better taxonomic assignment.

    In order to make sure that the primers won’t detect other major rice-associated bacteria, mostly pathogenic, we extended our in silico analysis to the type and pathotype strains of Burkholderia glumae, Pseudomonas fuscovaginae, P. oryzae, two species of Sphingomonas, and two pathovars of X. oryzae (Supplementary Table S2) (Ham et al. 2011; Kini et al. 2017c; Miyajima et al. 1983; Niño-Liu et al. 2006; Yu et al. 2013). In all cases, the number of mismatches exceeded what would be expected to allow DNA amplification, with at least 5 mismatches for atpD, 12 mismatches for gyrB, 8 mismatches for infB, and 13 mismatches for rpoB. Notably, at least one primer per primer pair had four mismatches (atpD), six mismatches (infB), or seven mismatches (gyrB and rpoB), thus preventing DNA amplification from these templates. We also tested a set of reference strains for these rice-pathogenic bacteria, and for none of them was any DNA amplification observed, except for the primer pair that amplifies a portion of the 16S rRNA gene (Supplementary Table S3).

    To reduce the costs and handling time, we generated a multiplex PCR scheme that can work with both purified genomic DNA and bacterial lysates. In both cases, sufficient specificity and sensitivity were obtained, allowing detection of as low as 0.5 ng of DNA or 104 CFU/ml for all three Pantoea spp. Notably, the scheme also allowed the diagnosis of the bacteria in seed samples. This simple scheme will be of particular interest for phytopathologists, especially in Africa and other less-developed regions. Indeed, diseases due to infections by Pantoea spp. appear to be emerging in Africa, as recently documented for Benin and Togo (Kini et al. 2017a, b). In this study, the presence of three plant-pathogenic Pantoea spp. has been demonstrated for 11 African countries. The fact that many BLB-like symptomatic rice samples contained Pantoea bacteria suggests that infection by Pantoea spp. is an underestimated source for BLB and might be widespread in Africa. However, more rigorous sampling schemes are required to determine the incidence and prevalence of Pantoea spp. in various rice-growing areas in Africa.

    Among the 607 Pantoea isolates, we detected 218 strains (36%; Supplementary Table S1) that could not be assigned to any of the three Pantoea spp. but likely belong to the genus Pantoea. This is an interesting observation that shows that the genus-specific primer pair not only serves as an internal positive control of the multiplex scheme but also has its own diagnostic value. Obviously, other species of Pantoea or closely related bacteria (e.g., species of the genus Mixta) are present in Africa and are likely to cause diseases of rice plants as well. However, it is still unknown whether or not this group of isolates contains other rice-pathogenic species. Pathogenicity assays need to confirm or disprove their status as novel pathogens. Future work using MLSA and whole-genome sequencing will address a subset of the isolates tested in this study.

    Acknowledgments

    We thank T. Afolabi (Africa Rice Center Cotonou, Benin) and S. Fabre and F. Auguy (IRD, Cirad, University Montpellier, IPME, Montpellier, France) for excellent technical support, and C. Tollenaere for testing this new diagnostic tool in the international laboratory “LMI Patho-Bios” (IRD-INERA Observatoire des Agents Phytopathogènes en Afrique de l’Ouest) in Burkina Faso and for helpful comments on the manuscript.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.

    Funding: Support was provided by the International Foundation for Science grant C/5921-1 and the Institute de Recherche pour le Développement Allocation de Recherche pour une Thèse au Sud.