First Vanilla planifolia High-Density Genetic Linkage Map Provides Quantitative Trait Loci for Resistance to Fusarium oxysporum.

Fusarium oxysporum f. sp. radicis-vanillae (Forv), the causal agent of root and stem rot disease, is the main pathogen affecting vanilla production. Sources of resistance have been reported in Vanilla planifolia G. Jackson ex Andrews, the main cultivated vanilla species. In this study, we developed the first high-density genetic map in this species with 1,804 genotyping-by-sequencing (GBS)-generated single nucleotide polymorphism (SNP) markers using 125 selfed progenies of the CR0040 traditional vanilla cultivar. Sixteen linkage groups (LG) were successfully constructed, with a mean of 113 SNPs and an average length of 207 cM per LG. The map had a high density with an average of 5.45 SNP every 10 cM and an average distance of 1.85 cM between adjacent markers. The first three LG were aligned against the first assembled chromosome of CR0040, and the other 13 LG were correctly associated with the other 13 assembled chromosomes. The population was challenged with the highly pathogenic Forv strain Fo072 using the root-dip inoculation method. Five traits were mapped, and 20 QTLs were associated with resistance to Fo072. Among the genes retrieved in the CR0040 physical regions associated with QTLs, genes potentially involved in biotic resistance mechanisms, coding for kinases, E3 ubiquitin ligases, pentatricopeptide repeat-containing proteins, and one leucine-rich repeat receptor underlying the qFo72_08.1 QTL have been highlighted. This study should provide useful resources for marker-assisted selection in V. planifolia.

Life history parameters and predation capacities of Nesidiocoris volucer: a new biological control agent for tomato crop.

Whiteflies are one of the major pests of tomato under greenhouses, and their control partly relies on biocontrol strategies. Among those biocontrol agents, parasitoids or predators are widely used. However, the introduction of a biocontrol agent in a new area is not trivial. For that reason, we investigated the use of a tropical native mirid, Nesidiocoris volucer (Hemiptera: Miridae), for the biological control of whiteflies among other insect pests on tomato crops under greenhouses in the subtropical island of La Réunion, France. Nesidiocoris volucer life history traits and plant injury were examined. Nymphs developed and survived between 15 and 30°C and required on average 49.41 days at 15°C and on average 10.50 days at 30°C to develop (nymph survival >94%). At 25°C, each female produced on average 65 eggs. Nesidiocoris volucer was able to feed on several prey species, but performed better on whiteflies than on spider mites or thrips. No N. volucer feeding injury was observed on tomato. Nesidiocoris volucer has also been found in tropical countries of Africa, and we believe that the data presented on this natural enemy could be of great importance for the biocontrol of whiteflies in tropical areas.

Joint species distributions reveal the combined effects of host plants, abiotic factors and species competition as drivers of species abundances in fruit flies.

The relative importance of ecological factors and species interactions for shaping species distributions is still debated. The realised niches of eight sympatric tephritid fruit flies were inferred from field abundance data using joint species distribution modelling and network inference, on the whole community and separately on three host plant groups. These estimates were then confronted the fundamental niches of seven fly species estimated through laboratory-measured fitnesses on host plants. Species abundances depended on host plants, followed by climatic factors, with a dose of competition between species sharing host plants. The relative importance of these factors mildly changed among the three host plant groups. Despite overlapping fundamental niches, specialists and generalists had almost distinct realised niches, with possible competitive exclusion of generalists by specialists on Cucurbitaceae. They had different assembly rules: Specialists were mainly influenced by their adaptation to host plants, while generalist abundances varied regardless of their fundamental host use.

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

BACKGROUND: Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. RESULTS: Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml- 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml- 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. CONCLUSIONS: We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

Host plant range of a fruit fly community (Diptera: Tephritidae): does fruit composition influence larval performance?

BACKGROUND: Phytophagous insects differ in their degree of specialisation on host plants, and range from strictly monophagous species that can develop on only one host plant to extremely polyphagous species that can develop on hundreds of plant species in many families. Nutritional compounds in host fruits affect several larval traits that may be related to adult fitness. In this study, we determined the relationship between fruit nutrient composition and the degree of host specialisation of seven of the eight tephritid species present in La Réunion; these species are known to have very different host ranges in natura. In the laboratory, larval survival, larval developmental time, and pupal weight were assessed on 22 fruit species occurring in La Réunion. In addition, data on fruit nutritional composition were obtained from existing databases. RESULTS: For each tephritid, the three larval traits were significantly affected by fruit species and the effects of fruits on larval traits differed among tephritids. As expected, the polyphagous species Bactrocera zonata, Ceratitis catoirii, C. rosa, and C. capitata were able to survive on a larger range of fruits than the oligophagous species Zeugodacus cucurbitae, Dacus demmerezi, and Neoceratitis cyanescens. Pupal weight was positively correlated with larval survival and was negatively correlated with developmental time for polyphagous species. Canonical correspondence analysis of the relationship between fruit nutrient composition and tephritid survival showed that polyphagous species survived better than oligophagous ones in fruits containing higher concentrations of carbohydrate, fibre, and lipid. CONCLUSION: Nutrient composition of host fruit at least partly explains the suitability of host fruits for larvae. Completed with female preferences experiments these results will increase our understanding of factors affecting tephritid host range.

Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

Frequency-dependent assistance as a way out of competitive exclusion between two strains of an emerging virus.

Biological invasions are the main causes of emerging viral diseases and they favour the co-occurrence of multiple species or strains in the same environment. Depending on the nature of the interaction, co-occurrence can lead to competitive exclusion or coexistence. The successive fortuitous introductions of two strains of Tomato yellow leaf curl virus (TYLCV-Mld and TYLCV-IL) in Réunion Island provided an ideal opportunity to study the invasion of, and competition between, these worldwide emerging pathogens. During a 7-year field survey, we observed a displacement of the resident TYLCV-Mld by the newcomer TYLCV-IL, with TYLCV-Mld remaining mostly in co-infected plants. To understand the factors associated with this partial displacement, biological traits related to fitness were measured. The better ecological aptitude of TYLCV-IL in single infections was demonstrated, which explains its rapid spread. However, we demonstrate that the relative fitness of virus strains can drastically change between single infections and co-infections. An epidemiological model parametrized with our experimental data predicts that the two strains will coexist in the long run through assistance by the fitter strain. This rare case of unilateral facilitation between two pathogens leads to frequency-dependent selection and maintenance of the less fit strain.

Virome release of an invasive exotic plant species in southern France.

The increase in human-mediated introduction of plant species to new regions has resulted in a rise of invasive exotic plant species (IEPS) that has had significant effects on biodiversity and ecosystem processes. One commonly accepted mechanism of invasions is that proposed by the enemy release hypothesis (ERH), which states that IEPS free from their native herbivores and natural enemies in new environments can outcompete indigenous species and become invasive. We here propose the virome release hypothesis (VRH) as a virus-centered variant of the conventional ERH that is only focused on enemies. The VRH predicts that vertically transmitted plant-associated viruses (PAV, encompassing phytoviruses and mycoviruses) should be co-introduced during the dissemination of the IEPS, while horizontally transmitted PAV of IEPS should be left behind or should not be locally transmitted in the introduced area due to a maladaptation of local vectors. To document the VRH, virome richness and composition as well as PAV prevalence, co-infection, host range, and transmission modes were compared between indigenous plant species and an invasive grass, cane bluestem (Bothriochloa barbinodis), in both its introduced range (southern France) and one area of its native range (Sonoran Desert, Arizona, USA). Contrary to the VRH, we show that invasive populations of B. barbinodis in France were not associated with a lower PAV prevalence or richness than native populations of B. barbinodis from the USA. However, comparison of virome compositions and network analyses further revealed more diverse and complex plant-virus interactions in the French ecosystem, with a significant richness of mycoviruses. Setting mycoviruses apart, only one putatively vertically transmitted phytovirus (belonging to the Amalgaviridae family) and one putatively horizontally transmitted phytovirus (belonging to the Geminiviridae family) were identified from B. barbinodis plants in the introduced area. Collectively, these characteristics of the B. barbinodis-associated PAV community in southern France suggest that a virome release phase may have immediately followed the introduction of B. barbinodis to France in the 1960s or 1970s, and that, since then, the invasive populations of this IEPS have already transitioned out of this virome release phase, and have started interacting with several local mycoviruses and a few local plant viruses.

Phylostems: a new graphical tool to investigate temporal signal of heterochronous sequences datasets.

Motivation: Molecular tip-dating of phylogenetic trees is a growing discipline that uses DNA sequences sampled at different points in time to co-estimate the timing of evolutionary events with rates of molecular evolution. Importantly, such inferences should only be performed on datasets displaying sufficient temporal signal, a feature important to test prior to any tip-dating inference. For this purpose, the most popular method considered to-date has been the 'root-to-tip regression' which consist in fitting a linear regression of the number of substitutions accumulated from the root to the tips of a phylogenetic tree as a function of sampling times. The main limitation of the regression method, in its current implementation, relies in the fact that the temporal signal can only be tested at the whole-tree scale (i.e. its root). Results: To overcome this limitation we introduce Phylostems, a new graphical user-friendly tool developed to investigate temporal signal within every clade of a phylogenetic tree. We provide a 'how to' guide by running Phylostems on an empirical dataset and supply guidance for results interpretation. Availability and implementation: Phylostems is freely available at https://pvbmt-apps.cirad.fr/apps/phylostems.

Herbarium specimen sequencing allows precise dating of Xanthomonas citri pv. citri diversification history.

Herbarium collections are an important source of dated, identified and preserved DNA, whose use in comparative genomics and phylogeography can shed light on the emergence and evolutionary history of plant pathogens. Here, we reconstruct 13 historical genomes of the bacterial crop pathogen Xanthomonas citri pv. citri (Xci) from infected Citrus herbarium specimens. Following authentication based on ancient DNA damage patterns, we compare them with a large set of modern genomes to estimate their phylogenetic relationships, pathogenicity-associated gene content and several evolutionary parameters. Our results indicate that Xci originated in Southern Asia ~11,500 years ago (perhaps in relation to Neolithic climate change and the development of agriculture) and diversified during the beginning of the 13th century, after Citrus diversification and before spreading to the rest of the world (probably via human-driven expansion of citriculture through early East-West trade and colonization).

Cage study of spinosad-based bait efficacy on Bactrocera cucurbitae, Dacus ciliatus, and Dacus demmerezi (Diptera: Tephritidae) in Reunion Island.

On Reunion Island, Bactrocera cucurbitae (Coquillett), Dacus ciliatus (Loew), and Dacus demmerezi (Bezzi) cause severe damage to Cucurbit crops. The aim of the study was to test in field cages the effectiveness of Synéis-appât (Dow AgroSciences), a spinosad-based bait (0.02% of spinosad) on both attraction and mortality of young adults (6-9 d old) of these three species. The effects of gender were also evaluated for all species whereas the effects of protein deprivation were tested with B. cucurbitae only. For the first 15 min after application, significantly more B. cucurbitae adults (21.7 +/- 1.8%) were attracted to the bait than D. demmerezi (7.6 +/- 2.4%) and D. ciliatus (2.7 +/- 1.4%); the subsequent response (30-75 min after bait application) of D. demmerezi was statistically similar to that recorded for B. cucurbitae; whereas the response ofD. ciliatus to the bait was consistently significantly lower. Adult mortality was significantly higher for B. cucurbitae (94.6 +/- 0.7%) than for D. demmerezi (85.7 +/- 2.1%), and was significantly higher for the latter than for D. ciliatus (60.4 +/- 4.4%). Sex had no significant effect on the mortality rate for each species. The efficiency of the bait was the same for B. cucurbitae adults regardless whether or not the diet included proteins. Overall, Synéis-appât appears to be more effective against B. cucurbitae and B. demmerezi than against D. ciliatus. In Reunion Island, this bait could constitute a useful component in the framework of Integrated Pest Management.

Impact of Special Drying Schemes on Color Stability of Mangoes with Different Maturity Degrees.

A previous study demonstrated that the color of 4 mm mango slices is altered very slightly by drying for 5 h at 60 °C, 30% RH and 1 m/s. The objectives of this complementary study were to determine the impact of various drying procedures encountered in the drying units on color alterations of sulfite-free mango slices from heterogeneous raw material due to variable maturity degrees of mangoes. Drying procedures with various temperature/humidity/duration combinations were performed to analyze their effects on the color of natural dried mangoes according to the degree of fruit maturity. They were dried at an air speed of 1.0 m/s for 5 h according to 3 schemes: standard drying (SD) at 60 °C and 30% RH; wet drying (WD) for 1 h at 60 °C and 60% RH, followed by 4 h SD; and finally, hot drying (HD) for 4 h SD, followed by 1 h at 80 °C and 30% RH. The color of the mango slices was analyzed before and after drying. SD preserves the color of fresh mangoes very well, whatever their maturity stage. A relatively slow drying onset corresponding to WD has a highly adverse impact, which becomes greater as the degree of maturity increases. There is already significant browning on mangoes with near-optimum quality (L* = 75; H* = 92). Applying high temperature at the end of the drying procedure (HD) for 20% of the time has a more limited adverse impact with immature mangoes that are the most sensitive. Linear regressions were assessed to represent the relationships of color differences between drying schemes according to mango maturity degrees. These statistical models showed a significant increase in color degradation in the case of WD and a decrease in color differences in the case of HD with the advance in fruit maturity.

The African citrus psyllid Trioza erytreae: An efficient vector of Candidatus Liberibacter asiaticus.

Introduction: Huanglonbing (HLB) is the most serious disease of citrus in the world, associated with three non-cultivable phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), Ca L. africanus (CLaf) and Ca L. americanus (CLam). CLas is transmitted by the Asian citrus psyllid Diaphorina citri, and has spread to several countries. The African psyllid Trioza erytreae, the vector of CLaf occurs in Africa and neighbouring islands. Only two major citrus-growing regions - Australia/New Zealand and the Mediterranean Basin - are still HLB-free in the world. However, T. erytreae has recently been introduced into continental Europe (Portugal and Spain) and has become a potential threat to citrus production. The transmission of CLas by T. erytreae had been postulated but never tested. To evaluate the risk of T. erytreae transmitting CLas, comparative transmissions of CLas by T. erytreae and D. citri were assessed. Methods: Transmission tests were performed on excised leaves and seedlings of Citrus volkameriana with different inoculation access periods (in series) for both insect species. Quantifications of bacterial titers were made in excised leaves, seedlings three and six months after inoculation and on individual insects. Results: Our results showed that T. erytreae was able to efficiently acquire CLas. Furthermore, T. erytreae carried significantly higher bacterial titers than D. citri, and was able to efficiently transmit the bacteria to seedlings at a similar rate that D. citri highlighting the high risk of spread of the most aggressive variant of HLB (CLas) by T. erytreae in Europe. Discussion: Thus, extreme precautions to prevent any entry of CLas into Europe should be adopted.

Synergy between an emerging monopartite begomovirus and a DNA-B component.

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

A novel synthetic quantification standard including virus and internal report targets: application for the detection and quantification of emerging begomoviruses on tomato.

BACKGROUND: Begomovirus is a genus of phytopathogenic single-stranded DNA viruses, transmitted by the whitefly Bemisia tabaci. This genus includes emerging and economically significant viruses such as those associated with Tomato Yellow Leaf Curl Disease, for which diagnostic tools are needed to prevent dispersion and new introductions. Five real-time PCRs with an internal tomato reporter gene were developed for accurate detection and quantification of monopartite begomoviruses, including two strains of the Tomato yellow leaf curl virus (TYLCV; Mld and IL strains), the Tomato leaf curl Comoros virus-like viruses (ToLCKMV-like viruses) and the two molecules of the bipartite Potato yellow mosaic virus. These diagnostic tools have a unique standard quantification, comprising the targeted viral and internal report amplicons. These duplex real-time PCRs were applied to artificially inoculated plants to monitor and compare their viral development. RESULTS: Real-time PCRs were optimized for accurate detection and quantification over a range of 2 × 10(9) to 2 × 10(3) copies of genomic viral DNA/µL for TYLCV-Mld, TYLCV-IL and PYMV-B and 2 × 10(8) to 2 × 10(3) copies of genomic viral DNA/µL for PYMV-A and ToLCKMV-like viruses. These real-time PCRs were applied to artificially inoculated plants and viral loads were compared at 10, 20 and 30 days post-inoculation. Different patterns of viral accumulation were observed between the bipartite and the monopartite begomoviruses. Interestingly, PYMV accumulated more viral DNA at each date for both genomic components compared to all the monopartite viruses. Also, PYMV reached its highest viral load at 10 dpi contrary to the other viruses (20 dpi). The accumulation kinetics of the two strains of emergent TYLCV differed from the ToLCKMV-like viruses in the higher quantities of viral DNA produced in the early phase of the infection and in the shorter time to reach this peak viral load. CONCLUSIONS: To detect and quantify a wide range of begomoviruses, five duplex real-time PCRs were developed in association with a novel strategy for the quantification standard. These assays should be of a great interest for breeding programs and epidemiological surveys to monitor viral populations.

Impact of Preharvest and Postharvest on Color Changes during Convective Drying of Mangoes.

The purpose of this study was to evaluate the impact of the harvest stage, ripening conditions and maturity on color changes of cv. 'Cogshall' and cv. 'Kent' variety mangoes during drying. A total of four harvests were undertaken, and the fruits were ripened at 20 and 35 °C for five different ripening times at each temperature. At each ripening time, mangoes were dried at 60 °C/30% RH/1.5 m/s for 5 h. A wide physico-chemical and color variability of fresh and dry pulp was created. The relationships according to the L*, H* and C* coordinates were established using mixed covariance regression models in relation to the above pre- and postharvest (preprocess) parameters. According to the L* coordinate results, browning during drying was not affected by the preprocess parameters. However, dried slices from mangoes ripened at 35 °C exhibited better retention of the initial chroma, and had a greater decrease in hue than dried slices from mangoes ripened at 20 °C. However, fresh mango color, successfully managed by the pre- and postharvest conditions, had more impact on dried mango color than the studied parameters. The preprocess parameters were effective levers for improving fresh mango color, and consequently dried mango color.

Pineapple Mycobiome Related to Fruitlet Core Rot Occurrence and the Influence of Fungal Species Dispersion Patterns.

Fruitlet Core Rot (FCR) is a fungal disease that negatively impacts the quality of pineapple, in particular the 'Queen Victoria' cultivar. The main FCR causal agent has been identified as Fusariumananatum. This study focused on the correlation between FCR disease occurrence, fungal diversity, and environmental factors. FCR incidence and fungal species repartition patterns were spatially contextualized with specific surrounding parameters of the experimental plots. The mycobiome composition of healthy and diseased fruitlets was compared in order to search for potential fungal markers. A total of 240 pineapple fruits were sampled, and 344 fungal isolates were identified as belonging to 49 species among 17 genera. FCR symptom distribution revealed a significant gradient that correlated to that of the most abundant fungal species. The association of wind direction and the position of proximal cultivated crops sharing pathogens constituted an elevated risk of FCR incidence. Five highly represented species were assayed by Koch's postulates, and their pathogenicity was confirmed. These novel pathogens belonging to Fusariumfujikuroi and Talaromycespurpureogenus species complexes were identified, unravelling the complexity of the FCR pathosystem and the difficulty of apprehending the pathogenesis over the last several decades. This study revealed that FCR is an airborne disease characterized by a multi-partite pathosystem.

Diversity and Geographical Structure of Xanthomonas citri pv. citri on Citrus in the South West Indian Ocean Region.

A thorough knowledge of genotypic and phenotypic variations (e.g., virulence, resistance to antimicrobial compounds) in bacteria causing plant disease outbreaks is key for optimizing disease surveillance and management. Using a comprehensive strain collection, tandem repeat-based genotyping techniques and pathogenicity assays, we characterized the diversity of X. citri pv. citri from the South West Indian Ocean (SWIO) region. Most strains belonged to the prevalent lineage 1 pathotype A that has a wide host range among rutaceous species. We report the first occurrence of genetically unrelated, nonepidemic lineage 4 pathotype A* (strains with a host range restricted to Mexican lime and related species) in Mauritius, Moheli and Réunion. Microsatellite data revealed that strains from the Seychelles were diverse, grouped in three different clusters not detected in the Comoros and the Mascarenes. Pathogenicity data suggested a higher aggressiveness of strains of one of these clusters on citron (Citrus medica). With the noticeable exception of the Comoros, there was no sign of recent interisland movement of the pathogen. Consistent with this finding, the copL gene, a marker for the plasmid-borne copLAB copper resistance that was recently identified in Réunion, was not detected in 568 strains from any islands in the SWIO region apart from Réunion.

Multiplex nested PCR for detection of Xanthomonas axonopodis pv. allii from onion seeds.

Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes (pilW and pilX) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444- to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 x 10(2) CFU g(-1) or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen.

Populations of Xanthomonas citri pv. mangiferaeindicae from asymptomatic mango leaves are primarily endophytic.

Epiphytic survival of several Xanthomonas pathovars has been reported, but most studies failed to determine whether such populations were resident epiphytes, resulting from latent infections, or casual epiphytes. This study aimed at understanding the nature of Xanthomonas citri pv. mangiferaeindicae populations associated with asymptomatic leaves. When spray-inoculated on mango leaves cv. Maison Rouge, the pathogen multiplied markedly in association with juvenile leaves, but was most often detected as low population sizes (<1 x 10(3) cfu g(-1)) in association with mature leaves. Our results suggest a very low biological significance of biofilm-associated populations of X. citri pv. mangiferaeindicae, while saprophytic microbiota associated with mango leaves survived frequently as biofilms. A chloroform vapor-based disinfestation assay which kills cells specifically located on the leaf surface and not those located within the leaf mesophyll was developed. When applied to spray-inoculated leaves maintained under controlled environmental conditions, 155 out of the 168 analyzed datasets collected over three assessment dates for seven bacterial strains representative of the genetic diversity of the pathogen failed to demonstrate a significant X. citri pv. mangiferaeindicae population decrease on chloroform treated leaves up to 13 days after inoculation. We conclude that an efficient survival of X. citri pv. mangiferaeindicae present on mango leaf surfaces following a limited dissemination event is largely dependent on the availability of juvenile plant tissues. The bacterium gains access to protected sites (e.g., mesophyll) through stomata where it becomes endophytic and eventually causes disease. Chloroform vapor-based disinfestation assays should be useful for further studies aiming at evaluating survival sites of bacteria associated with the phyllosphere.