The Rice ILI2 Locus Is a Bidirectional Target of the African Xanthomonas oryzae pv. oryzae Major Transcription Activator-like Effector TalC but Does Not Contribute to Disease Susceptibility.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

Confirmation report of Bacterial Leaf Streak disease of rice caused by Xanthomonas oryzae pv. oryzicola in Senegal.

Xanthomonas oryzae pv. X. oryzicola (Xoc), the causal agent of Bacterial Leaf Streak (BLS), is considered as one of the most important emerging pathogens of rice in Africa. This disease is estimated as responsible of 20 to 30% yield loss (Sileshi et Gebeyehu 2021) and has been characterized in several west African countries including Mali and Burkina Faso since 2003 and more recently in Ivory Coast (Wonni et al. 2014, Diallo et al. 2021). Presence of BLS symptoms in Senegal were reported by Trinh in 1980 but, to our knowledge, BLS occurrence has never been validated further and no strain of Xoc have ever been isolated from Senegalese rice fields. Xoc is transmitted by seeds which contribute to its spread through the rice trade (Sileshi et Gebeyehu 2021). To confirm Trinh's observations, we surveyed rice fields between 2014 and 2016 in eight different regions where rice is produced in Senegal. Typical disease symptoms characterized by yellow-brown to black translucent leaf streaks sometimes along with exudates, were detected in fields of several regions and collected. Leaf pieces were successively sanitized, rinsed in sterile water, and symptomatic fragments were ground using the Qiagen Tissue Lyser System (QIAGEN, Courtaboeuf, France). The leaf powder was diluted in 1.5 ml of sterile water and incubated for 30 minutes at room temperature. Ten µl of the suspension was streaked on semi-selective PSA medium and incubated at 28°C for 3 to 7 days. Characteristic round, convex, mucous, straw-yellow Xoc candidate colonies were purified from six individual leaf samples from three distinct sites in the northern Senegal River Valley. To confirm their identity, isolated strains were tested for pathogenicity and molecular characterization. All isolates were subjected to the multiplex PCR developed for the identification of X. oryzae pathovars (Lang et al., 2010) and revealed the same PCR profile (two amplicons of 324 and 691 base pairs) similar to that of the Xoc reference strain BLS256. Leaves of 5-week-old plants of O. sativa cv. Kitaake were infiltrated with a needleless syringe containing a bacterial suspension set at an optical density of 0.5. Upon seven days of incubation under greenhouse conditions (27 ± 1°C with a 12-hour photoperiod), all infiltrated spots (2 spots on 3 plants per isolate) developed water-soaked lesions similar to those caused by control strain BLS256, except when leaves were infiltrated with water. Symptomatic leaf tissues were ground and plated on PSA medium, resulting in colonies with typical Xanthomonas morphology that were diagnosed as Xoc by multiplex PCR typing, thus fulfilling Koch's postulate. At last, four of the isolates were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F and XgyrB1R (Young et al., 2008). Analysis of 780bp partial gyrB sequences of strains S18-3-4, S23-1-12, S52-1-4 and S52-1-10 highlighted 100% identity with the gyrB sequence of strain BLS256 (Acc. No. CP003057). To our knowledge, this is the first report of BLS in Senegal which is supported by molecular characterization methods. This study validates the presence of BLS in Senegal and will serve as a basis for future efforts of rice breeding for locally adapted resistance. More studies are needed to clarify the spatial distribution and prevalence of BLS in Senegal as rice cultivation is expanding rapidly in the country.

Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae.

Xanthomonas phaseoli pv. manihotis (Xpm) and X. cassavae (Xc) are two bacterial pathogens attacking cassava. Cassava bacterial blight (CBB) is a systemic disease caused by Xpm, which might have dramatic effects on plant growth and crop production. Cassava bacterial necrosis is a nonvascular disease caused by Xc with foliar symptoms similar to CBB, but its impacts on the plant vigour and the crop are limited. In this review, we describe the epidemiology and ecology of the two pathogens, the impacts and management of the diseases, and the main research achievements for each pathosystem. Because Xc data are sparse, our main focus is on Xpm and CBB.

Resistance and susceptibility QTL identified in a rice MAGIC population by screening with a minor-effect virulence factor from Xanthomonas oryzae pv. oryzae.

Effective and durable disease resistance for bacterial blight (BB) of rice is a continuous challenge due to the evolution and adaptation of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), on cultivated rice varieties. Fundamental to this pathogens' virulence is transcription activator-like (TAL) effectors that activate transcription of host genes and contribute differently to pathogen virulence, fitness or both. Host plant resistance is predicted to be more durable if directed at strategic virulence factors that impact both pathogen virulence and fitness. We characterized Tal7b, a minor-effect virulence factor that contributes incrementally to pathogen virulence in rice, is a fitness factor to the pathogen and is widely present in geographically diverse strains of Xoo. To identify sources of resistance to this conserved effector, we used a highly virulent strain carrying a plasmid borne copy of Tal7b to screen an indica multi-parent advanced generation inter-cross (MAGIC) population. Of 18 QTL revealed by genome-wide association studies and interval mapping analysis, six were specific to Tal7b (qBB-tal7b). Overall, 150 predicted Tal7b gene targets overlapped with qBB-tal7b QTL. Of these, 21 showed polymorphisms in the predicted effector binding element (EBE) site and 23 lost the EBE sequence altogether. Inoculation and bioinformatics studies suggest that the Tal7b target in one of the Tal7b-specific QTL, qBB-tal7b-8, is a disease susceptibility gene and that the resistance mechanism for this locus may be through loss of susceptibility. Our work demonstrates that minor-effect virulence factors significantly contribute to disease and provide a potential new approach to identify effective disease resistance.

Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles.

Vascular plant pathogens travel long distances through host veins, leading to life-threatening, systemic infections. In contrast, nonvascular pathogens remain restricted to infection sites, triggering localized symptom development. The contrasting features of vascular and nonvascular diseases suggest distinct etiologies, but the basis for each remains unclear. Here, we show that the hydrolase CbsA acts as a phenotypic switch between vascular and nonvascular plant pathogenesis. cbsA was enriched in genomes of vascular phytopathogenic bacteria in the family Xanthomonadaceae and absent in most nonvascular species. CbsA expression allowed nonvascular Xanthomonas to cause vascular blight, while cbsA mutagenesis resulted in reduction of vascular or enhanced nonvascular symptom development. Phylogenetic hypothesis testing further revealed that cbsA was lost in multiple nonvascular lineages and more recently gained by some vascular subgroups, suggesting that vascular pathogenesis is ancestral. Our results overall demonstrate how the gain and loss of single loci can facilitate the evolution of complex ecological traits.

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems.

Xanthomonas oryzae (Xo) are globally important rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the United States have been well characterized. Xanthomonas campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali, and Uganda to determine the genetic relatedness of this organism with Xo. Novel transcription activator-like effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the Leersia perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity (ANI), type III (T3) effector repertoires, and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.

Functional and Genome Sequence-Driven Characterization of tal Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice.

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene.

Allelic variation for broad-spectrum resistance and susceptibility to bacterial pathogens identified in a rice MAGIC population.

Quantitative trait loci (QTL) that confer broad-spectrum resistance (BSR), or resistance that is effective against multiple and diverse plant pathogens, have been elusive targets of crop breeding programmes. Multiparent advanced generation intercross (MAGIC) populations, with their diverse genetic composition and high levels of recombination, are potential resources for the identification of QTL for BSR. In this study, a rice MAGIC population was used to map QTL conferring BSR to two major rice diseases, bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pathovars (pv.) oryzicola (Xoc) and oryzae (Xoo), respectively. Controlling these diseases is particularly important in sub-Saharan Africa, where no sources of BSR are currently available in deployed varieties. The MAGIC founders and lines were genotyped by sequencing and phenotyped in the greenhouse and field by inoculation with multiple strains of Xoc and Xoo. A combination of genomewide association studies (GWAS) and interval mapping analyses revealed 11 BSR QTL, effective against both diseases, and three pathovar-specific QTL. The most promising BSR QTL (qXO-2-1, qXO-4-1 and qXO-11-2) conferred resistance to more than nine Xoc and Xoo strains. GWAS detected 369 significant SNP markers with distinguishable phenotypic effects, allowing the identification of alleles conferring disease resistance and susceptibility. The BSR and susceptibility QTL will improve our understanding of the mechanisms of both resistance and susceptibility in the long term and will be immediately useful resources for rice breeding programmes.

The overexpression of RXam1, a cassava gene coding for an RLK, confers disease resistance to Xanthomonas axonopodis pv. manihotis.

MAIN CONCLUSION: The overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136, suggesting that RXam1 might be implicated in strain-specific resistance. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is a prevalent disease in all regions, where cassava is cultivated. CBB is a foliar and vascular disease usually controlled through host resistance. Previous studies have found QTLs explaining resistance to several Xam strains. Interestingly, one QTL called XM5 that explained 13% of resistance to XamCIO136 was associated with a similar fragment of the rice Xa21-resistance gene called PCR250. In this study, we aimed to further identify and characterize this fragment and its role in resistance to CBB. Screening and hybridization of a BAC library using the molecular marker PCR250 as a probe led to the identification of a receptor-like kinase similar to Xa21 and were called RXam1 (Resistance to Xam 1). Here, we report the functional characterization of susceptible cassava plants overexpressing RXam1. Our results indicated that the overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136. This suggests that RXAM1 might be implicated in strain-specific resistance to XamCIO136.

RNA-Seq analysis reveals insight into enhanced rice Xa7-mediated bacterial blight resistance at high temperature.

Plant disease is a major challenge to agriculture worldwide, and it is exacerbated by abiotic environmental factors. During some plant-pathogen interactions, heat stress allows pathogens to overcome host resistance, a phenomenon which could severely impact crop productivity considering the global warming trends associated with climate change. Despite the importance of this phenomenon, little is known about the underlying molecular mechanisms. To better understand host plant responses during simultaneous heat and pathogen stress, we conducted a transcriptomics experiment for rice plants (cultivar IRBB61) containing Xa7, a bacterial blight disease resistance (R) gene, that were infected with Xanthomonas oryzae, the bacterial blight pathogen of rice, during high temperature stress. Xa7-mediated resistance is unusual relative to resistance mediated by other R genes in that it functions better at high temperatures. Using RNA-Seq technology, we identified 8,499 differentially expressed genes as temperature responsive in rice cultivar IRBB61 experiencing susceptible and resistant interactions across three time points. Notably, genes in the plant hormone abscisic acid biosynthesis and response pathways were up-regulated by high temperature in both mock-treated plants and plants experiencing a susceptible interaction and were suppressed by high temperature in plants exhibiting Xa7-mediated resistance. Genes responsive to salicylic acid, an important plant hormone for disease resistance, were down-regulated by high temperature during both the susceptible and resistant interactions, suggesting that enhanced Xa7-mediated resistance at high temperature is not dependent on salicylic acid signaling. A DNA sequence motif similar to known abscisic acid-responsive cis-regulatory elements was identified in the promoter region upstream of genes up-regulated in susceptible but down-regulated in resistant interactions. The results of our study suggest that the plant hormone abscisic acid is an important node for cross-talk between plant transcriptional response pathways to high temperature stress and pathogen attack. Genes in this pathway represent an important focus for future study to determine how plants evolved to deal with simultaneous abiotic and biotic stresses.

Characterization of the Xanthomonas translucens Complex Using Draft Genomes, Comparative Genomics, Phylogenetic Analysis, and Diagnostic LAMP Assays.

Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA4 , an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (> 90%), but absent in the other major X. oryzae pathovar. These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice.

Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas.

How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL effectors with diverse predicted targets and is effective against African strains of Xanthomonas oryzae pv. oryzicola.

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes.

Identification of novel major and minor QTLs associated with Xanthomonas oryzae pv. oryzae (African strains) resistance in rice (Oryza sativa L.).

BACKGROUND: Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of Bacterial Leaf Blight (BB), an emerging disease in rice in West-Africa which can induce up to 50 % of yield losses. So far, no specific resistance gene or QTL to African Xoo were mapped. The objectives of this study were to identify and map novels and specific resistance QTLs to African Xoo strains. RESULTS: The reference recombinant inbred lines (RIL) mapping population derived from the cross between IR64 and Azucena was used to investigate Xoo resistance. Resistance to African and Philippine Xoo strains representing different races was assessed on the RIL population under greenhouse conditions. Five major quantitative trait loci (QTL) for resistance against African Xoo were located on different chromosomes. Loci on chromosomes 1, 7, 9, 10 and 11 explained as much as 13 %, 37 %, 13 %, 11 % and 15 % of resistance variation, respectively. A major novel QTL located on chromosome 7 explained 37 % of the phenotypic variance to the African Xoo corresponding to race A3 whereas that on chromosome 11 is effective to all African races tested. Together with genes and QTLs for resistance to bacterial blight previously described, the QTLs described here were mapped onto the reference O. sativa subs japonica (var. Nipponbare) physical map. CONCLUSION: We characterized new resistance QTLs. While some co-localize with known resistance genes/QTLs to Asian strains, others are specific to African strains. We result with new information on genes and QTLs for resistance to bacterial blight that will be useful for controlling the disease.

Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.

Four bacterial strains, designated M89, M92, M97(T), and M106, were isolated in a previous study from surface-sterilized leaves of rice (Oryza sativa) or murainagrass (Ischaemum rugosum) at three sites in Mali, Africa. Here they were examined by a polyphasic taxonomic approach and analysis of a whole-genome sequence. Phylogenetic analyses based on 16S rRNA sequence and multilocus sequence analysis of seven genes showed that these four strains formed a distinct lineage representing a novel species within the genus Xanthomonas. This was supported by whole-genome average nucleotide identity values calculated from comparisons of strain M97(T) with established Xanthomonas species. The strains can be differentiated from the known Xanthomonas species on the basis of their fatty acid and carbohydrate utilization profiles. Population growth studies on rice confirmed that these bacteria multiply in rice leaves without causing symptoms. Identification of this novel species can be accomplished by using diagnostic primer sets or by gyrB gene sequence analysis. We propose to classify these rice- and grass-associated bacteria as Xanthomonas maliensis sp. nov. with strain M97(T) = CFBP7942(T) = LMG27592(T) as the type strain.

A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage.

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification.

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10(4) to 10(5) CFU ml(-1), while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.

Development of a Genomics-Based LAMP (Loop-Mediated Isothermal Amplification) Assay for Detection of Pseudomonas fuscovaginae from Rice.

The vast amount of data available through next-generation sequencing technology is facilitating the design of diagnostic marker systems. This study reports the use of draft genome sequences from the bacterial plant pathogen Pseudomonas fuscovaginae, the cause of sheath brown rot of rice, to describe the genetic diversity within a worldwide collection of strains representing the species. Based on a comparative analysis with the draft sequences, primers for a loop-mediated isothermal amplification (LAMP) assay were developed to identify P. fuscovaginae. The assay reported here reliably differentiated strains of P. fuscovaginae isolated from rice from a range of other bacteria that are commonly isolated from rice and other plants using a primer combination designated Pf8. The LAMP assay identified P. fuscovaginae purified DNA, live or heat-killed cells from pure cultures, and detected the bacterium in extracts or exudates from infected host plant material. The P. fuscovaginae LAMP assay is a suitable diagnostic tool for the glasshouse and laboratory and could be further developed for in-field surveys.