A new multi locus variable number of tandem repeat analysis scheme for epidemiological surveillance of Xanthomonas vasicola pv. musacearum, the plant pathogen causing bacterial wilt on banana and enset

Xanthomonas vasicola pv. musacearum (Xvm) which causes Xanthomonas wilt (XW) on banana (Musa accuminata x balbisiana) and enset (Ensete ventricosum), is closely related to the species Xanthomonas vasicola that contains the pathovars vasculorum (Xvv) and holcicola (Xvh), respectively pathogenic to sugarcane and sorghum. Xvm is considered a monomorphic bacterium whose intra-pathovar diversity remains poorly understood. With the sudden emergence of Xvm within east and central Africa coupled with the unknown origin of one of the two sublineages suggested for Xvm, attention has shifted to adapting technologies that focus on identifying the origin and distribution of the genetic diversity within this pathogen. Although microbiological and conventional molecular diagnostics have been useful in pathogen identification. Recent advances have ushered in an era of genomic epidemiology that aids in characterizing monomorphic pathogens. To unravel the origin and pathways of the recent emergence of XW in Eastern and Central Africa, there was a need for a genotyping tool adapted for molecular epidemiology. Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA) is able to resolve the evolutionary patterns and invasion routes of a pathogen. In this study, we identified microsatellite loci from nine published Xvm genome sequences. Of the 36 detected microsatellite loci, 21 were selected for primer design and 19 determined to be highly typeable, specific, reproducible and polymorphic with two- to four- alleles per locus on a sub-collection. The 19 markers were multiplexed and applied to genotype 335 Xvm strains isolated from seven countries over several years. The microsatellite markers grouped the Xvm collection into three clusters; with two similar to the SNP-based sublineages 1 and 2 and a new cluster 3, revealing an unknown diversity in Ethiopia. Five of the 19 markers had alleles present in both Xvm and Xanthomonas vasicola pathovars holcicola and vasculorum, supporting the phylogenetic closeliness of these three pathovars. Thank to the public availability of the haplotypes on the MLVABank database, this highly reliable and polymorphic genotyping tool can be further used in a transnational surveillance network to monitor the spread and evolution of XW throughout Africa.. It will inform and guide management of Xvm both in banana-based and enset-based cropping systems. Due to the suitability of MLVA-19 markers for population genetic analyses, this genotyping tool will also be used in future microevolution studies

A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences

Xanthomonas vasicola pv. musacearum (Xvm) is a bacterial pathogen responsible for the economically important Xanthomonas wilt disease on banana and enset crops in Sub-Saharan Africa. Given that the symptoms are similar to those of other diseases, molecular diagnosis is essential to unambiguously identify this pathogen and distinguish it from closely related strains not pathogenic on these hosts. Currently, Xvm identification is based on polymerase chain reaction (PCR) with GspDm primers, targeting the gene encoding general secretory protein D. Experimental results and examination of genomic sequences revealed poor specificity of the GspDm PCR. Here, we present and validate five new Xvm-specific primers amplifying only Xvm strains

Risks Posed by Intercrops and Weeds as Alternative Hosts to Xanthomonas campestris pv. musacearum in Banana Fields

Alternative host plants are important in the survival and perpetuation of several crop pathogens and have been suspected to play a role in the survival of Xanthomonas campestris pv. musacearum (Xcm) and perpetuation of Xanthomonas wilt (XW) disease of banana and enset. This study determined the potential risk posed by two weeds (Canna spp. and wild sorghum) and common banana intercrops (maize, millet, sorghum, taro, and sugarcane) as alternative hosts to Xcm. The study employed screenhouse experiments, laboratory procedures and diagnosis of banana fields in XW-affected landscapes. Typical XW symptoms were only observed in artificially inoculated Canna sp., with an incidence of 96%. Leaf lesions characteristic of xanthomonads occurred on millet (50%) and sorghum (35%), though the plants recovered. No symptoms occurred in maize, sugarcane, taro or wild sorghum. However, Xcm was recovered from all these plant species, with higher recoveries in Canna sp. (47%), millet (27%), sugarcane (27%), and wild sorghum (25%). Only isolates recovered from Canna sp., millet, sorghum and wild sorghum caused disease in banana plantlets. The presence and incidence of XW on-farm was positively associated with the presence of susceptible ABB Musa genotypes and negatively with number of banana cultivars on farm and household access to training on XW management. Only 0.02% of field sampled Canna spp. plants had Xcm. Risk posed by Canna spp. on-farm could be limited to tool transmission as it has persistent floral bracts that prevent insect-mediated infections. Given the high susceptibility, perennial nature and propagation through rhizomes of Canna sp., it could pose a moderate-high risk, thus warranting some attention in the management of XW disease. Sugarcane could offer a low-moderate risk due to its perennial nature and propagation through rhizomes while risk from maize, millet, and sorghum was deemed zero-low due to their annual nature, wind-mediated mode of pollination and propagation through seed. Understanding the interactions of a crop pathogen with other plants is thus important when diversifying agroecosystems. The study findings also suggest other factors such as cultivar composition and management of the disease at farm and landscape level to be important in the perpetuation of XW disease

SNP-based genotyping and whole-genome sequencing reveal previously unknown genetic diversity in Xanthomonas vasicola pv. musacearum, causal agent of banana xanthomonas wilt, in its presumed Ethiopian origin

For decades, Xanthomonas vasicola pv. musacearum (Xvm) has been an economically important bacterial pathogen on enset in Ethiopia. Since 2001, Xvm has also been responsible for significant losses to banana crops in several East and Central African countries, with devastating consequences for smallholder farmers. Understanding the genetic diversity within Xvm populations is essential for the smart design of transnationally reasoned, durable, and effective management practices. Previous studies have revealed limited genetic diversity in Xvm, with East African isolates from banana each falling into one of two closely related clades previously designated as sublineages SL 1 and SL 2, the former of which had also been detected on banana and enset in Ethiopia. Given the presumed origin of Xvm in Ethiopia, we hypothesized that both clades might be found in that country, along with additional genotypes not seen in Central and East African bananas. Genotyping of 97 isolates and whole-genome sequencing of 15 isolates revealed not only the presence of SL 2 in Ethiopia, but additional diversity beyond SL 1 and SL 2 in four new clades. Moreover, SL 2 was detected in the Democratic Republic of Congo, where previously SL 1 was the only clade reported. These results demonstrate a greater range of genetic diversity among Xvm isolates than previously reported, especially in Ethiopia, and further support the hypothesis that the East/Central Africa xanthomonas wilt epidemic has been caused by a restricted set of genotypes drawn from a highly diverse pathogen pool in Ethiopia

Transfer of Xanthomonas campestris pv. arecae, and Xanthomonas campestris pv. musacearum to Xanthomonas vasicola (Vauterin) as Xanthomonas vasicola pv. arecae comb. nov., and Xanthomonas vasicola pv. musacearum comb. nov. and description of Xanthomonas vasicola pv. vasculorum pv. nov.

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 29,79-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events

A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences

Xanthomonas vasicola pv. musacearum (Xvm) is a bacterial pathogen responsible for the economically important Xanthomonas wilt disease on banana and enset crops in Sub-Saharan Africa. Given that the symptoms are similar to those of other diseases, molecular diagnosis is essential to unambiguously identify this pathogen and distinguish it from closely related strains not pathogenic on these hosts. Currently, Xvm identification is based on polymerase chain reaction (PCR) with GspDm primers, targeting the gene encoding general secretory protein D. Experimental results and examination of genomic sequences revealed poor specificity of the GspDm PCR. Here, we present and validate five new Xvmspecific primers amplifying only Xvm strains.Agropolis Fondation under the reference ID 1605-025 through the « Investissements d’avenir » program (Labex Agro: ANR-10-LABX-0001-01), under the frame of I-SITE MUSE (ANR-16-IDEX-0006). Additional financial support came from the CRP-Roots Tubers and Banana.https://www.heliyon.comam2019Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

Risks posed by intercrops and weeds as alternative hosts to xanthomonas campestris pv. Musacearum in Banana Fields

Alternative host plants are important in the survival and perpetuation of several crop pathogens and have been suspected to play a role in the survival of Xanthomonas campestris pv. musacearum (Xcm) and perpetuation of Xanthomonas wilt (XW) disease of banana and enset. This study determined the potential risk posed by two weeds (Canna spp. and wild sorghum) and common banana intercrops (maize, millet, sorghum, taro, and sugarcane) as alternative hosts to Xcm. The study employed screenhouse experiments, laboratory procedures and diagnosis of banana fields in XW-affected landscapes. Typical XW symptoms were only observed in artificially inoculated Canna sp., with an incidence of 96%. Leaf lesions characteristic of xanthomonads occurred on millet (50%) and sorghum (35%), though the plants recovered. No symptoms occurred in maize, sugarcane, taro or wild sorghum. However, Xcm was recovered from all these plant species, with higher recoveries in Canna sp. (47%), millet (27%), sugarcane (27%), and wild sorghum (25%). Only isolates recovered from Canna sp., millet, sorghum and wild sorghum caused disease in banana plantlets. The presence and incidence of XW on-farm was positively associated with the presence of susceptible ABB Musa genotypes and negatively with number of banana cultivars on farm and household access to training on XW management. Only 0.02% of field sampled Canna spp. plants had Xcm. Risk posed by Canna spp. on-farm could be limited to tool transmission as it has persistent floral bracts that prevent insect-mediated infections. Given the high susceptibility, perennial nature and propagation through rhizomes of Canna sp., it could pose a moderate-high risk, thus warranting some attention in the management of XW disease. Sugarcane could offer a low-moderate risk due to its perennial nature and propagation through rhizomes while risk from maize, millet, and sorghum was deemed zero-low due to their annual nature, wind-mediated mode of pollination and propagation through seed. Understanding the interactions of a crop pathogen with other plants is thus important when diversifying agroecosystems. The study findings also suggest other factors such as cultivar composition and management of the disease at farm and landscape level to be important in the perpetuation of XW disease.</p

Influence of altitude as a proxy for temperature on key Musa pests and diseases in watershed areas of Burundi and Rwanda

Pests and diseases are key biotic constraints limiting banana production among smallholder farmers in Eastern and Central Africa. Climate changemay favour pest and disease development and further exacerbate the vulnerability of smallholder farming systems to biotic constraints. Information on effects of climate change on pests and pathogens of banana is required by policy makers and researchers in designing control strategies and adaptation plans. Since altitude is inversely related to temperature, this study used the occurrence of key banana pests and diseases along an altitude gradient as a proxy for the potential impact of changes in temperature associated with global warming on pests and diseases. We assessed the occurrence of banana pests and diseases in 93 banana fields across three altitude ranges in Burundi and 99 fields distributed in two altitude ranges in Rwanda watersheds. Incidence and prevalence of Banana Bunchy Top Disease (BBTD) and Fusarium wilt (FW) was significantly associated with temperature and altitude in Burundi, revealing that increasing temperatures may lead to upward movement of banana diseases. No significant associations with temperature and altitude were observed for weevils, nematodes and Xanthomonas wilt of banana (BXW). Data collected in this study provides a baseline to verify and guide modelling work to predict future pest and disease distribution according to climate change scenarios. Such information is useful in informing policy makers and designing appropriate management strategies

Sources of resistance in Musa to Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt

It is claimed that, with the exception of Musa balbisiana, all banana varieties are susceptible to bacterial wilt caused by Xanthomonas campestris pv. musacearum (Xcm). Despite being resistant to Xcm infection, M. balbisiana is not preferred for breeding because it belongs to the BB genome subgroup, while most edible bananas are of the A genome. To identify potential sources of resistance to Xcm, 72 banana accessions representing the Musa genetic diversity were evaluated in an outdoor confined potted trial. The midribs of the youngest leaf of 3-month-old banana plants were inoculated with 108 CFU mL 1 of Xcm isolate USY13P, and symptom development assessed weekly for 4 months. Results confirmed that M. balbisiana genotypes are indeed resistant to Xcm. Varieties within the Musa acuminata subsp. zebrina (AA) set were further identified as potentially useful sources of Xcm resistance. These findings reveal the potential to develop banana and plantain varieties with tolerance to Xcm.RTB-CRPhttps://onlinelibrary.wiley.com/journal/13653059am2019Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog