Analysis of Xanthomonas oryzae pv. oryzicola population in Mali and Burkina Faso reveals a high level of genetic and pathogenic diversity.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola was first reported in Africa in the 1980s. Recently, a substantial reemergence of this disease was observed in West Africa. Samples were collected at various sites in five and three different rice-growing regions of Burkina Faso and Mali, respectively. Sixty-seven X. oryzae pv. oryzicola strains were isolated from cultivated and wild rice varieties and from weeds showing BLS symptoms. X. oryzae pv. oryzicola strains were evaluated for virulence on rice and showed high variation in lesion length on a susceptible cultivar. X. oryzae pv. oryzicola strains were further characterized by multilocus sequence analysis (MLSA) using six housekeeping genes. Inferred dendrograms clearly indicated different groups among X. oryzae pv. oryzicola strains. Restriction fragment length polymorphism analysis using the transcriptional activator like effector avrXa7 as probe resulted in the identification of 18 haplotypes. Polymerase chain reaction-based analyses of two conserved type III effector (T3E) genes (xopAJ and xopW) differentiated the strains into distinct groups, with xopAJ not detected in most African X. oryzae pv. oryzicola strains. XopAJ functionality was confirmed by leaf infiltration on 'Kitaake' rice Rxo1 lines. Sequence analysis of xopW revealed four groups among X. oryzae pv. oryzicola strains. Distribution of 43 T3E genes shows variation in a subset of X. oryzae pv. oryzicola strains. Together, our results show that African X. oryzae pv. oryzicola strains are diverse and rapidly evolving, with a group endemic to Africa and another one that may have evolved from an Asian strain.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Uganda.

In June 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the booting stage in the Doho rice irrigation scheme, Butaleja district, and at the tillering stage in Nambale, Iganga district and Magada, Namutumba district of Uganda. In areas surveyed, disease incidence was about 80, 40, and 30% in Doho, Nambale, and Magada, respectively. Outside the irrigation schemes, it was lower but widespread. Affected leaves showed typical BLS symptoms, such as water-soaked lesions, translucent stripes, and yellow-brown to black streaks, sometimes with visible exudates at the leaf surfaces. To check for the presence of the bacteria, symptomatic leaves were ground in sterile water and the suspension obtained was subjected to a multiplex PCR assay for X. oryzae pathovars, leading to the three diagnostic DNA fragments for X. oryzae pv. oryzicola (3). In parallel, bacterial strains were isolated from surface-sterilized symptomatic leaves. To this end, rice leaves were ground in sterile distilled water and serial dilutions of the cell suspensions were plated on semi-selective PSA medium (4). Each of the three samples yielded yellow, mucoid Xanthomonas-like colonies that resembled the positive control strain MAI10 (1). These isolates were named Ug_1, Ug_10, and Ug_14, which originated from Doho, Magada, and Nambale, respectively. Multiplex PCR on the pure cultures strongly supported that these isolates corresponded to X. oryzae pv. oryzicola. Two isolates, Ug_1 and Ug_14, were further subjected to partial DNA sequence analysis of the gyrB gene upon PCR amplification using the primers XgyrB1F and XgyrB1R (5). The 467-bp DNA sequence was identical to the gyrB sequences from the X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), and MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of strain Ug_1 was submitted to GenBank (KJ921786). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cultivars Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. For this purpose, bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water at a concentration of 1 × 108 CFU/ml. Bacterial suspensions were sprayed on leaves of rice seedlings. Four seedlings per accession and isolate were inoculated. Fifteen days after incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), inoculated leaves exhibited typical water-soaked lesions with yellow exudates that were similar to the symptoms seen in the fields. Re-isolation of the bacteria from the diseased leaves yielded colonies with the typical morphology of Xanthomonas. Multiplex PCR and sequence analysis of portions of the gyrB gene confirmed that these isolates are X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. One of the three isolates, Ug_1, has been deposited in the Collection Française de Bactéries Phytopathogènes (CFBP) as strain CFBP 8171 ( http://www.angers-nantes.inra.fr/cfbp/ ). Further surveys and strain collections in East and Central Africa will help assess the geographic distribution and importance of BLS. References: (1) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 108 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5'-ACGAGTACAACCCGGACAA-3') and XgyrB1R (5'-CCCATCARGGTGCTGAAGAT-3'). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99A of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola. References: (1) I. W. Buddenhagen. Int. Rice Comm. Newsl. 34:74, 1985. (2) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) D. O. Niño-Liu et al. Mol. Plant Pathol. 7:303, 2006. (5) I. Wonni et al. Plant Dis. 95:72, 2011.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi.

On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Genetic Structure and TALome Analysis Highlight a High Level of Diversity in Burkinabe Xanthomonas Oryzae pv. oryzae Populations.

Bacterial Leaf Blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat for food security in many rice growing countries including Burkina Faso, where the disease was first reported in the 1980's. In line with the intensification of rice cultivation in West-Africa, BLB incidence has been rising for the last 15 years. West-African strains of Xoo differ from their Asian counterparts as they (i) are genetically distant, (ii) belong to new races and, (iii) contain reduced repertoires of Transcription Activator Like (TAL) effector genes. In order to investigate the evolutionary dynamics of Xoo populations in Burkina Faso, 177 strains were collected from 2003 to 2018 in three regions where BLB is occurring. Multilocus VNTR Analysis (MLVA-14) targeting 10 polymorphic loci discriminated 24 haplotypes and showed that Xoo populations were structured according to their geographical localization and year of collection. Considering their major role in Xoo pathogenicity, we assessed the TAL effector repertoires of the 177 strains upon RFLP-based profiling. Surprisingly, an important diversity was revealed with up to eight different RFLP patterns. Finally, comparing neutral vs. tal effector gene diversity allowed to suggest scenarios underlying the evolutionary dynamics of Xoo populations in Burkina Faso, which is key to rationally guide the deployment of durably resistant rice varieties against BLB in the country.

Efficient enrichment cloning of TAL effector genes from Xanthomonas.

Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on •genome-informed in silico optimization of restriction digestions,•selective restriction digestion of genomic DNA, and•size fractionation of DNA fragments. Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.

Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper.

The AvrBs3 protein of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria is targeted to host-plant cells by the bacterial Hrp type III secretion system. In pepper plants containing the Bs3 resistance gene, AvrBs3 induces the hypersensitive response (HR). AvrBs3 recognition is thought to occur in the plant cell nucleus as HR induction is dependent on nuclear localization signals (NLSs) and an acidic transcription activation domain (AAD). In a search for AvrBs3-interacting pepper proteins using the yeast two-hybrid system, we have isolated eight different classes of cDNA inserts including two genes for importin alpha proteins. Importin alpha is part of the nuclear import machinery and interacts with AvrBs3 through an NLS in the carboxy-terminus of the protein, both in yeast and in vitro. The mechanism of AvrBs3 recognition was further studied by analysis of the C-terminal AAD. This putative transcription-activation domain was shown to be required for AvrBs3 HR-inducing activity, and could be functionally replaced with the VP16 AAD from the Herpes simplex virus. Our data support the model in which the AvrBs3 effector localizes to the nucleus, where the Bs3-mediated surveillance system of resistant plants detects AvrBs3 through its interference with host gene transcription.

How the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria conquers the host.

Abstract Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper and tomato. Pathogenicity on susceptible plants and the induction of the hypersensitive reaction (HR) on resistant plants requires a number of genes, designated hrp, most of which are clustered in a 23-kb chromosomal region. Nine hrp genes encode components of a type III protein secretion apparatus that is conserved in Gram-negative plant and animal pathogenic bacteria. We have recently demonstrated that Xcv secretes proteins into the culture medium in a hrp-dependent manner. Substrates of the Hrp secretion machinery are pathogenicity factors and avirulence proteins, e.g. AvrBs3. The AvrBs3 protein governs recognition, i.e. HR induction, when bacteria infect pepper plants carrying the corresponding resistance gene Bs3. Intriguingly, the AvrBs3 protein contains eukaryotic signatures such as nuclear localization signals (NLS), and has been shown to act inside the plant cell. We postulate that AvrBs3 is transferred into the plant cell via the Hrp type III pathway and that recognition of AvrBs3 takes place in the plant cell nucleus.