Development of two loop-mediated isothermal amplification (LAMP) genomics-informed diagnostic protocols for rapid detection of Pantoea species on rice.

At least three species of Pantoea are responsible for bacterial blight disease and grain discoloration of rice in Sub-Saharan Africa. Thus, measures need to be taken to limit the pathogens' dispersion and robust diagnostic tools are required for rapid and cheap diagnosis in the field as well as for routine seed certification or control. Therefore, several diagnostic tools such as simplex and multiplex PCR schemes and a semi-selective medium have been developed and are being used. However, the use of these tools is time-consuming, expensive and therefore limited to laboratories that can afford the chemicals. We have therefore developed two isothermal loop amplification (LAMP) protocols, one of which detects all Pantoea species in the genus and another one that is specific for P. ananatis.•The novel LAMP assays allow rapid and sensitive detection of these bacteria.•They will help plant protection services in routine field and laboratory tests especially for monitoring the phytosanitary status of rice seeds.

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

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.

Structure of African Populations of Pyricularia oryzae from Rice.

Rice blast, caused by the filamentous ascomycete Pyricularia oryzae, is one of the most devastating diseases of rice. Four genetic clusters were previously identified, and three have a large geographic distribution. Asia is the center of diversity and the origin of most migrations to other continents, and sexual reproduction persisted only in the South China-Laos-North Thailand region, which was identified as the putative center of origin of all P. oryzae populations on rice. Despite the importance of rice blast disease, little is known about the diversity and the population structure of the pathogen in Africa (including Madagascar). The present study was intended to describe the structure of African populations of P. oryzae and identify the relationship between African and worldwide genetic clusters. A set of 2,057 strains (937 African and 1,120 Madagascan strains) were genotyped with 12 simple sequence repeat markers to assess the diversity and the population structure of P. oryzae. Four genetic clusters were identified in Africa and Madagascar. All four clusters previously identified are present in Africa. Populations from West Africa, East Africa, and Madagascar are highly differentiated. The geographic structure is consistent with limited dispersion and with some migration events between neighboring countries. The two mating types are present in Africa with a dominance of Mat1.2, but no female-fertile strain was detected, supporting the absence of sexual reproduction on this continent. This study showed an unsuspected high level of genetic diversity of P. oryzae in Africa and suggested several independent introductions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Resources of Three West African Strains of Pantoea ananatis Causing Bacterial Blight and Grain Discoloration of Rice.

Members of the genus Pantoea have been reported as pathogens for many economically important crops, including rice. Little is known about their host-pathogen interactions at the molecular level and the lack of comprehensive genome data impedes targeted breeding strategies toward resistant rice cultivars. Here, we describe the structural and functional annotation of the draft genome sequences of three rice-pathogenic Pantoea ananatis strains, ARC272, ARC310, and ARC311, which were isolated in Burkina Faso, Togo, and Benin, respectively. The genome sequences of these strains will help in developing molecular diagnostic tools and provide new insight into common traits that may enable P. ananatis to infect rice.

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.

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.

An Efficient Inoculation Technique to Assess the Pathogenicity of Pantoea Species Associated to Bacterial Blight of Rice.

Bacteria blight diseases of rice due to several genera of pathogenic bacteria are one of the major constraints worldwide for rice production. The disease can be best managed through host plant resistance sources. For most of these bacteria such as Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae, specific diagnostic techniques that include molecular and pathogenicity tests have been developed. However, for Pantoea spp., information on pathogenicity assay is very limited and protocols used are not uniform. Most authors use the leaf clipping method. In this paper, we describe the protocol for mechanical inoculation of rice seedlings aged 35 days. The method consists of infiltrating bacterial suspensions at concentrations of 108 CFU/ml, with a needleless syringe into the intercellular and interveinal spaces of rice leaves underside at about 4-5 cm below the leaf tip. This method can be used for a standardized pathogenicity assessment, germplasm resistance evaluation for identifying and characterizing resistance sources.

Mycotoxin concentrations in rice from three climatic locations in Africa as affected by grain quality, production site, and storage duration.

Information on the mycotoxin contamination of rice in Africa is limited although the risk of contamination is high. In this study, domestic milled rice processed by actors using suboptimal methods was purchased and total fumonisin (FUM), zearalenone, and aflatoxin concentrations determined at 0, 90, and 180 days after storage. Three different climatic locations, Cotonou (Benin) in the Guinea savanna, Yaoundé (Cameroon) in the Tropical forest, and N'diaye (Senegal) in the Sahel, were selected as storage sites. Subsets of the samples collected from Glazoue (Benin), Ndop (Cameroon), and Dagana (Senegal) were stored in plastic woven bags under room conditions in the respective sites with or without calcium oxide (burnt scallop shell-BSS, 0.1% w/w) treatment. Multivariance analysis showed that FUM concentration was positively influenced by the duration of storage only while zearalenone concentration was negatively influenced by relative humidity and head rice but positively by impurities. Zearalenone concentration was also influenced by sample collection/storage location, processing type, and duration of storage. Aflatoxin concentration was influenced negatively by storage room temperature and head rice but positively by impurities and chalky grains. In addition, aflatoxin concentration was influenced by collection/storage location and processing type. BSS treatment followed by storage for 6 months had no effect on the concentration of the three assessed mycotoxins. Strategies to reduce the risk of mycotoxin contamination in study sites will include the improvement of physical rice quality through better pre- and postharvest practices and proper packaging of both treated rice and untreated rice in hermetic systems before marketing and storage.

Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings.

Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation - one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).

Identification of a Hypervirulent Pathotype of Rice yellow mottle virus: A Threat to Genetic Resistance Deployment in West-Central Africa.

Rice yellow mottle virus (RYMV) causes high losses to rice production in Africa. Several sources of varietal high resistance are available but the emergence of virulent pathotypes that are able to overcome one or two resistance alleles can sometimes occur. Both resistance spectra and viral adaptability have to be taken into account to develop sustainable rice breeding strategies against RYMV. In this study, we extended previous resistance spectrum analyses by testing the rymv1-4 and rymv1-5 alleles that are carried by the rice accessions Tog5438 and Tog5674, respectively, against isolates that are representative of RYMV genetic and pathogenic diversity. Our study revealed a hypervirulent pathotype, named thereafter pathotype T', that is able to overcome all known sources of high resistance. This pathotype, which is spatially localized in West-Central Africa, appears to be more abundant than previously suspected. To better understand the adaptive processes of pathotype T', molecular determinants of resistance breakdown were identified via Sanger sequencing and validated through directed mutagenesis of an infectious clone. These analyses confirmed the key role of convergent nonsynonymous substitutions in the central part of the viral genome-linked protein to overcome RYMV1-mediated resistance. In addition, deep-sequencing analyses revealed that resistance breakdown does not always coincide with fixed mutations. Actually, virulence mutations that are present in a small proportion of the virus population can be sufficient for resistance breakdown. Considering the spatial distribution of RYMV strains in Africa and their ability to overcome the RYMV resistance genes and alleles, we established a resistance-breaking risk map to optimize strategies for the deployment of sustainable and resistant rice lines in Africa.

Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima.

KEY MESSAGE: A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene. Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.

Genetic variation in blast resistance in rice germplasm from West Africa.

The genetic variation in resistance to blast (Pyricularia oryzae Cavara) in 195 rice accessions comprising 3 species of the AA genome complex (Asian rice [Oryza sativa L.], African rice [Oryza glaberrima Steud.] and wild rice [Oryza barthii]) was investigated based on their patterns of reaction to standard differential blast isolates (SDBIs) and SSR marker polymorphism data. Cluster analysis of the polymorphism data of 61 SSR markers identified 3 major clusters: cluster A (mainly Japonica Group or upland accessions), cluster B (mainly Indica Group or lowland accessions) and cluster C (O. glaberrima and O. barthii). The accessions were classified again into 3 resistance groups based on reactions to SDBIs: group Ia (susceptible), group Ib (middle resistance) and group II (high resistance). Group Ia included only a few differential varieties, susceptible controls and the Japonica Group cultivar Nipponbare. Accessions in clusters A and B included all 3 resistance groups and showed a wide variation in blast resistance, but cluster C contained only group Ib. These results demonstrated that variations in Asian rice (O. sativa) accessions in West Africa were skewed toward high resistance and that variations in O. glaberrima and O. barthii were limited and lower than the Asian rice accessions.

Evidence that DL-3-aminobutyric acid and acibenzolar-S-methyl induce resistance against bacterial head rot disease of broccoli.

Head rot of broccoli caused by Pseudomonas marginalis (Brown) Stevens and P. fluorescens Migula is a major disease in Brittany (France). To date, no accession with a satisfactory field resistance has been identified, and available pesticides are not effective in controlling the disease. The aim of this study was to test whether acibenzolar-S-methyl (ASM), DL-3-aminobutyric acid (BABA) and potassium phosphonate (K(2)HPO(3)), known to induce resistance against various diseases, can help protect broccoli against head rot. The susceptible broccoli F1 hybrids Marathon and Shogun were grown in a greenhouse until head formation. They were then sprayed with ASM (0.23 mM AI), BABA (20 mM AI) or potassium phosphonate (37.41 mM AI) until runoff. In one experiment, heads from treated plants were excised, inoculated (10(4) cfu ml(-1)) and incubated in Magenta GA7 vessels. In another experiment, heads were inoculated on treated living plants. Disease ratings were made 5 days after inoculation. Antibiotic- and water-treated plants served as controls. Results obtained showed that, on excised treated heads, potassium phosphonate was not protective and disease scores were comparable with those of the water control. BABA- and ASM-treated excised heads were poorly, but significantly, protected. On whole plants with heads attached, the latter two compounds were much more effective. ASM-induced resistance increased in effectiveness over 8 days after inoculation, whilst that induced by BABA decreased. This result suggests that testing disease resistance inducers on excised broccoli heads is not accurate. ASM and BABA may offer alternative methods for controlling head rot of broccoli.