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

Abstract

Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by μl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety.

DNA Markers for Detection and Genotyping of Xanthomonas euroxanthea

“…When assessing the PCR detection limit of each marker individually, it was observed that XEA5 lowered the limit of detection value to 10 pg/µL, indicating that the sensitivity of the simplex PCR targeting XEA5 marker is ten-fold higher than the multiplex PCR. The detection limit of 0.1 ng/µL attained for the multiplex PCR is similar to what has been reported in other studies (0.02 and 0.5 ng/µL) [40,41].…”

Plant Growth-Promoting Bacteria as an Emerging Tool to Manage Bacterial Rice Pathogens

“…This conveys a sense of continuing excitement in the field of molecular plant pathology to control bacterial rice pathogens. To date, the main approaches to control bacterial rice pathogens include the production of disease-resistant rice varieties [ 34 , 35 , 36 ]; modification in cultural practices [ 37 , 38 ]; use of natural products or botanical extracts [ 39 , 40 , 41 ]; use of conventional and non-conventional chemicals [ 42 , 43 ]; coevolution analysis of the pathogen virulence and the host resistance genes [ 44 , 45 ]; transcriptomic analysis of pathogen along rice development [ 46 , 47 ]; improvement of diagnostic tools in the field for early detection of infectious diseases [ 48 ].…”

The impact of the rice production system (irrigated vs lowland) on root-associated microbiome from farmer's fields in western Burkina Faso

“…All three remained rare, with only one sample containing each of these sequences. The presence of B. glumae was described in Burkina Faso (but with no molecular data; Ouedraogo et al 2004), and targeted detection performed in two sites failed to detect B. glumae and A. avenae (Bangratz et al 2020). For fungal pathogens, we found ASVs assigned to ten genera comprising rice pathogens, including Bipolaris, Curvularia, Fusarium and Rhizoctonia.…”