Identification of genes involved in epiphytic survival of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Xanthomonas albilineans is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, at least some strains of the pathogen are spread by aerial means and are able to colonize the phyllosphere before entering the host plant. The objective of this study was to identify molecular factors that are involved in the epiphytic phase of the disease. Several wild-type strains of X. albilineans and a related non pathogenic species, as well as putative pathogenicity mutants of X. albilineans, were studied for their capacity to survive on sugarcane leaves. Four week-old tissue-cultured plantlets of cultivar CP68-1026 were immersed in bacterial suspensions at 107 cfu/ml. Foliar imprints of the plantlets were performed on selective growth medium 14 days after inoculation. The wild-type strain of X. albilineans that was not associated with aerial transmission of the pathogen, and that belongs to genetic group PFGE-A, had a lower epiphytical survival capacity than the wild-type strains that are aerially transmitted and that belong to genetic group PFGE-B. An outer-membrane A (XaOmpA1) mutant and surface polysaccharide mutants completely lost their capacity to survive on the leaf surface. In contrast, a motility mutant, a mutant affected in production of small molecules, and the non pathogenic species related to X. albilineans were all able to colonize the phyllosphere similarly to wild-type strains that are transmitted by aerial means. Mutants of the rpf gene cluster, involved in bacterial communication, showed different behaviors depending on the mutated gene. (Texte intégral

Development of the loop mediated isothermal amplification (LAMP) method for detection of Sugarcane yellow leaf virus

Sugarcane yellow leaf is a disease caused by Sugarcane yellow leaf virus (SCYLV). It is a major emerging disease of sugarcane that has been reported in numerous locations including Brazil, Mauritius, Reunion Island, French West Indies, South Africa, Swaziland, Malawi and Zimbabwe. The development of efficient tools to diagnose this disease is important, especially diagnostic methods that are effective both at the laboratory and at field level. This study aims at developing a rapid, sensitive and accurate method for the detection of SCYLV, using the Loop Mediated Isothermal Amplification (LAMP) technology. The presence of SCYLV was tested in sugarcane leaf samples by both tissue blot immunoassay (TBIA) and LAMP method. The efficiency of the LAMP technology to detect SCYLV in plants that were grown in CIRAD's quarantine greenhouses and in leaves that were collected from several locations including Reunion Island, Kenya and the French West Indies will be reported. (Texte intégral

Leaf scald of sugarcane

L'échaudure des feuilles de la canne à sucre est une maladie bactérienne vasculaire provoquée par Xanthomonas albilineans (Ashby) Dawson. Les conséquences économiques peuvent être graves lorsque des variétés sensibles sont atteintes dans les zones de production. La symptomatologie comporte une forme chronique, une forme aiguë ainsi que des phases dites de latence et d'éclipse qui rendent le diagnostic délicat. Actuellement, cette maladie est présente dans au moins 61 zones de production de la canne à sucre. Elle se propage d'autant plus rapidement qu'elle est transmise par les boutures. Les méthodes de lutte sont préventives : sélection variétale de plantes résistantes; contrôle des échanges de matériel végétal par une station de quarantaine; production de boutures parfaitement saines à partir des pépinières; techniques de thermothérapie des boutures. (Résumé d'auteur

L'échaudure des feuilles de la canne à sucre

L'échaudure des feuilles de la canne à sucre est une maladie bactérienne vasculaire provoquée par Xanthomonas albilineans (Ashby) Dawson. Les conséquences économiques peuvent être graves lorsque des variétés sensibles sont atteintes dans les zones de production. La symptomatologie comporte une forme chronique, une forme aiguë ainsi que des phases dites de latence et d'éclipse qui rendent le diagnostic délicat. Actuellement, cette maladie est présente dans au moins 61 zones de production de la canne à sucre. Elle se propage d'autant plus rapidement qu'elle est transmise par les boutures. Les méthodes de lutte sont préventives : sélection variétale de plantes résistantes; contrôle des échanges de matériel végétal par une station de quarantaine; production de boutures parfaitement saines à partir des pépinières; techniques de thermothérapie des bouture

Identification of viruses in Gramineae growing in the neighbourhood of CIRAD's sugarcane quarantine in Montpellier, France

CIRAD's sugarcane quarantine imports sugarcane varieties from different countries all over the world. These varieties, imported as cuttings or tissue-cultured plantlets, are likely to harbour pathogenic organisms and especially viruses. Among the 10 virus species that have or might have been introduced into CIRAD's quarantine, Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV) and Sugarcane yellow leaf virus (SCYLV) are the most liable to be spread outside the quarantine glasshouses. The risk of spread is due to the presence of insect vectors (in particular aphids such as Rhopalosiphum maydis) in the Mediterranean area and a large variety of weeds (mainly Gramineae) that are listed as alternate host plants of these viruses. Eighty-seven leaf samples (4 from maize, 10 from Johnson grass, 72 from sorghum and 1 from Setaria adhaerens) were collected in the neighbourhood of CIRAD's quarantine and tested for viruses using RT-PCR and RFLP techniques. Almost all samples showed mosaic symptoms and 86 were infected by Maize dwarf mosaic virus (MDMV) (SrMV-SCH cannot be excluded) and/or SCMV belonging to the monophyletic maize group. None of these viruses was likely to come from CIRAD's quarantine glasshouses. Neither SCMV belonging to the sugarcane group nor SCSMV was detected. A virus related to (but different from) SCYLV was detected in two Johnson grass samples, but its identity and origin have not been investigated yet. (Texte intégral

Visacane, an innovative quarantine tool for the exchange of pest and disease-free sugarcane germplasm

Sugarcane varietal improvement can not exclusively rely upon exchange and introduction of genetic resources via true seeds (fuzz). It also requires the introduction of vegetative propagation material (cuttings, tissue-cultured plantlets). The continued increase in international and intercontinental trade in plants has led to the enforcement of quarantine measures before introduction into a country because many plant pathogens can be carried and transmitted by vegetatively propagated material. Visacane is the new name of Cirad's sugarcane quarantine (http://visacane.cirad.fr/en/). It covers three main quarantine procedures: detection of pests and pathogens, elimination of pests and pathogens, and transfer of plant material free of pests and pathogens. It has been devoted to sugarcane quarantining for several decades. Besides phytosanitary constraints, Visacane takes also into account legal constraints and ensures, through appropriate contracts, that plant breeders' intellectual property rights over the transferred material are respected. Unlike most sugarcane quarantines that are essentially used to import sugarcane germplasm into a country, Visacane can import and export varieties from and to most sugarcane growing countries in the world, ensuring that the material is free from any important pest and disease causing pathogen. Until recently, the sugarcane quarantine process was aimed at detecting known pathogens harbored by the plant material and eliminating these pathogens whenever possible. It is an a priori process, because it only takes into account the pathogens that have been previously described and for which efficient detection tools exist. During the last three decades, several new viruses infecting sugarcane have been discovered, including Sugarcane bacilliform virus, Sugarcane yellow leaf virus, Sugarcane streak mosaic virus and the virus associated with Ramu stunt. In addition, the etiology of chlorotic streak, a disease known since 1929, has not been elucidated so far, although there is evidence for its infectious nature. Therefore, it can be assumed that unknown pathogens are still to be discovered in sugarcane, especially if these pathogens do not cause symptoms that can be easily observed. For these reasons, the research team associated with Visacane is setting up a new strategy of diagnostics, the so-called sequence-independent approach which aims at deciphering the virome (= the genomes of all the viruses that inhabit a particular organism). We believe that our forthcoming combined process, that will include our traditional approaches in addition to the metagenomics approach, will drastically improve our routine quarantine diagnostics. (Texte intégral

Modern tools for safe detection of diseases in sugarcane quarantine

Aims: Use of modern molecular tools in disease detection, different detection methods, visual observation, serological tests, PCR or RT-PCR tests have been described for elimination of pests and diseases from infected sugarcane germplasm. Application of strict quarantine measures to control movement of sugarcane germplasm is advocated. Methods and Results: Improvement of sugar crops relies, to a large extent, upon the cultivation of new sugarcane varieties that are either bred locally or imported from other geographical locations. In either case, there is a need for importing sugarcane germplasm from abroad. Because sugarcane is vegetatively propagated (stem cuttings, tissue-cultured plantlets), there is a high risk of introducing infectious diseases or pests from countries of origin. Therefore, strict quarantine measures must be applied to control movement of sugarcane germplasm. In the past, quarantine procedures relied almost exclusively upon the cultivation of imported varieties in isolated and closed premises (greenhouses), and the access to these areas was limited to a few duly authorized persons. Additionally, visual search for disease symptoms was performed during the entire growth cycle of the plants. These procedures are still in use at present time, but a wide range of tools for detecting and controlling diseases have been developed during the last decades, resulting in quarantine practices that are much safer and reliable. Nowadays, most of the efforts must be focused on the detection and elimination of symptomless or latent diseases such as leaf scald (Xanthomonas albilineans), ratoon stunting (Leifsonia xyli subsp. xyli) or yellow leaf (Sugarcane yellow leaf virus or SCYLV). Additionally, special attention must be paid to emerging diseases, the symptoms of which are sometimes rather unusual or difficult to detect, and for which efficient detection tools are not always available. Finally, detection tools must be very efficient in detecting low populations of the pathogen as well as all variants of this pathogen. Within the last decade, CIRAD's sugarcane quarantine had to face two emerging diseases, streak mosaic (Sugarcane streak mosaic virus or SCSMV) and yellow leaf. SCSMV appeared to be a heterogeneous virus, and none of the antisera used allowed us to detect all the isolates of our collection. Fortunately, an RT-PCR test developed in our laboratory has been quite sensitive and efficient. Since the end of the 1990s, sugarcane yellow leaf has been routinely detected in our quarantine using a tissue blot immuno-assay (TBIA) and RT-PCR tests. However, recent studies of the genetic diversity of SCYLV showed that some isolates of this virus were not systematically detected, and new and more universal primers were designed. A similar situation was experienced for the detection of sugarcane mosaic, a disease caused by two variable viruses: Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV). Following genetic diversity studies of SCMV, including virus isolates from various geographical origins, new primer pairs were designed and used in RT-PCR. Significance of study: Even though modem molecular tools have greatly improved disease detection in sugarcane quarantine, their exclusive use is not advisable. Very often, a combination of different detection methods must be performed: visual observation of the plants at several growth stages, isolation of bacterial pathogens on selective media, serological tests, PCR or RT-PCR tests. Additionally, the use of physical (hot water or hot air), chemical (fungicide and pesticide) treatments and apical meristem culture allow the elimination of many pests and diseases from infected sugarcane germplasm. (Texte intégral

Metagenomic screening of the sugarcane virome in Florida. [P.36]

Viral metagenomics has revolutionized the way pathologists decipher viral diseases. While the impact of this new approach is still debatable in plant virus diagnostics, viral metagenomics has already produced key advances in viral ecology and has the potential to become a central approach for viral surveillance at the ecosystem scale. A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013/2014. One hundred and eighty sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Sequence-independent next generation sequencing (NGS) of virion-associated nucleic acids (VANA) was used for detection and identification of viruses present within the collected leaf samples. All four previously reported sugarcane viruses occuring in Florida were detected: Sugarcane yellow leaf virus (149 infected samples out of 180), Sugarcane mosaic virus (2/180), Sugarcane mild mosaic virus (10/180) and Sugarcane bacilliform virus (51/180). Interestingly, this viral metagenomics approach also resulted in the detection of potential new viruses of sugarcane, including Chrysovirus, Mastrevirus, and Umbravirus. This study provided a snapshot vision of the SCYLV genetic diversity in 2013/2014 in Florida where several genotypes of this virus are present. It also allowed us to assemble the whole genome of at least one new mastrevirus species. (Résumé d'auteur