A diagnostic multiplex PCR scheme for identification of plant-associated bacteria of the genusPantoea

Abstract

12 13 Background: The genus Pantoea forms a complex of more than 25 species, among which 14 several cause diseases of several crop plants, including rice. Notably, strains of Pantoea 15 ananatis and Pantoea stewartii have been found to cause bacterial leaf blight of rice in Togo 16 and Benin, while other authors have observed that Pantoea agglomerans can also cause 17 bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to 18 plant diseases and yield losses of crop plants is currently not well documented, partly due to 19 the lack of efficient diagnostic tools. 20 21 Result: Using 34 whole genome sequences of the three-major plant-pathogenic Pantoea 22species, a set of PCR primers that specifically detect each of the three species, 23 P. agglomerans, P. ananatis, and P. stewartii, was designed. A multiplex PCR protocol which 24 can distinguish these three species and also detects members of other Pantoea species was 25 2 further developed. Upon validation on a set of reference strains, 609 suspected Pantoea 26 strains that were isolated from rice leaves or seeds originating from 11 African countries were 27 screened. In total, 41 P. agglomerans strains from eight countries, 79 P. ananatis strains from 28 nine countries, 269 P. stewartii strains from nine countries and 220 unsolved Pantoea strains 29 from ten countries were identified. The PCR protocol allowed detecting Pantoea bacteria 30 grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 5 ng/mL 31 of total genomic DNA and 1×10 5 CFU/mL of heated cells. 32 33 Conclusion: This new molecular diagnostic tool will help accurately diagnose major plant-34 pathogenic species of Pantoea. Due to its robustness, specificity, sensitivity, and cost 35 efficiency it will be very useful for plant protection services and for the epidemiological 36 surveillance of these important crop-threatening bacteria. 37 38 39 41 3 Background 42 43 The genus Pantoea was first described in 1989 and was recently taxonomically classified as a 44 member of the Erwiniaceae family [1]. More than 25 species of this genus have been 45 described and reported worldwide [2,3]. Etymologically, the genus name Pantoea is derived 46 from the Greek word 'Pantoios', which means "of all sorts or sources" and reflects the diverse 47 geographical and ecological sources from which the bacteria have been isolated. Several 48 species of the genus are qualified as versatile and ubiquitous bacteria because they have been 49 isolated from many different ecological niches and hosts [2,4]. Remarkably, some species 50 have the ability to colonize and interact with members of both the plant and the animal 51 Kingdom [5]. Among the plant-interacting species, Pantoea ananatis, Pantoea agglomerans 52 and Pantoea stewartii are well known for their phytopathogenic characteristics. They are 53 recognized as the causal agent of several diseases, such as leaf blight, spot disease, dieback, 54 grain discoloration, seed stalk rot, center rot, stem necros...

Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso

“…All this makes this protocol easy-to-use in different labs including those located in low-income countries. With a focus on the major bacterial rice diseases found in Africa, in particular the recently described Pantoea and Sphingomonas spp., this tool will complement already available detection protocols [31][32][33][34][35] for improved diagnostics of bacterial rice diseases and epidemiological surveillance in the field. Table 3.…”

“…For the design of Pantoea spp. specific primers, we targeted a region in the ATP synthase subunit beta AtpD (atpD) gene, which had been exploited for a diagnostic multiplex PCR scheme for three species of plant-pathogenic Pantoea species [32]. Similarly, Sphingomonas- PLOS ONE specific primers were designed for the 16S rRNA gene, which had been used for diagnosis of several Sphingomonas species on symptomatic rice leaf samples from eight West African countries [14].…”

“…In particular, efficient detection protocols are available for X. oryzae [31] and for Pantoea spp. [32]. Few recently introduced methods can simultaneously detect several rice pathogens.…”

Microbial Diversity and Metabolite Profile of Fermenting Millet in the Production of Hausa koko, a Ghanaian Fermented Cereal Porridge

“…The grain samples, used as the raw material to produce the Hausa koko, recorded more OTUs and higher relative abundance of the genus Pantoea compared to the other time points. This genus of Gram-negative bacteria of the Enterobacteriaceae family is usually isolated from several sources and ecological niches including plants (Kini et al, 2018;Azizi et al, 2020) and they are most probably associated with the original plant material and the soil environment (Sarita and Singh, 2016). The grain samples were also dominated by other Gram-negative microbes including Pseudomonas, Enterobacteriaceae, and Escherichia-Shigella, some of which may be potential pathogens and are commonly associated with feces, soil, and intestinal tracts of humans and warm-blooded animals and may cause gastroenteritis, diarrhea, vomiting, and nausea (Gadaga et al, 2004(Gadaga et al, , 2008.…”

A semi-selective medium to isolate and identify bacteria of the genus Pantoea

“…The characteristics of all the isolates used are given in Table 1 of the supplemental data file. Strains of Sphingomonas and Pantoea from the AfricaRice strain bank were diagnosed using, respectively, a monoplex and a Multiplex PCR scheme developed by Kini et al (2017aKini et al ( , 2018.…”