Detection of Sugarcane yellow leaf virus by a novel reverse transcription loop-mediated isothermal amplification method from three sugarcane production regions in Kenya

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 worldwide the last two decades. Efficient tools have been developed for detecting SCYLV but their use requires sophisticated facilities and still remains expensive. Our partners from developing countries cannot easily use them, which hamper an efficient and early diagnostic of the disease in sugarcane production regions. The main goal of this study was to develop an "easy-to-use" method for detecting SCYLV at the level of partner laboratories and potentially at the field level. We develop a novel reverse transcription loop-mediated isothermal amplification method (RTLAMP) for detecting SCYLV. This method was compared to the classical methods routinely used at CIRAD Montpellier sugarcane quarantine facilities (RT-PCR and Tissue Blot Immunoassay). The three methods were then used for detecting SCYLV in three sugarcane production regions from Kenya, for which no data of prevalence of the disease existed so far. The first results showed that the RT-LAMP is more efficient than the reference methods. SCYLV is present in the three sugarcane production regions with prevalence rate ranging from 5% to 20%. This is the first report of sugarcane yellow leaf disease in Kenya. Furthermore, this is the first report on the application of the LAMP assay for early diagnostic of sugarcane yellow leaf disease from sugarcane production regions. Due to its simplicity, sensitivity and cost-effectiveness for common use, we believe that this assay should be used as an early diagnostic tool by our partners at the field level. (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

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

Development of a multigenic metabarcoding of prokaryotic microbiota: application to Bacteria and Archaea of agronomical interest encountered in the rice rhizosphere of Camargue

It is our hypothesis that soil and plant microbiota are playing an eminent role in shaping both natural ecosystems and agricultural production systems. It is now widely admitted that bacterial communities show a great diversity in the rhizosphere and in the endosphere as endophytes. These microbial communities affect the fitness of both the hosts and the microbes and further play important roles in C and N cycles. Much less is known about the role played (if any) by archaea within both the plant rhizosphere and endosphere. Interestingly, a recent study has revealed a relatively high archaeal concentration in internal plant tissues (Müller et al., 2015). The objective of our study is to develop a multigenic metabarcoding approach to accurately describe the biodiversity of relevant microbial communities interacting with rice paddies in the Rhône delta region. This pilot agro-ecosystem was selected because it is annually subjected to several abiotic stresses (water stress associated to rice paddies flooding, soil salinity, etc.) that are likely to enhance archaeal growth. We will retain universal prokaryotic primers and archaeal specific primers within the 16S rRNA gene, two housekeeping genes, gyrB(encoding the ß subunit of the DNA gyrase) and rpoB (encoding the ß subunit of the RNA polymerase) for bacteria, and the thermosome TF55 gene (encoding a type II chaperonin) for archaea. Primers evaluation will be conducted on mock cultures and by sampling rice plants during both rice paddies flooding and drying periods. This study is likely to be a first step towards better understanding the dynamics of Camargue rice paddies microbial communities in relation with abiotic constraints

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

Assessment and characterization of the genetic diversity of viruses infecting cultivated yams (Dioscorea spp.) in Haïti

Several viral species are known to infect yams (Dioscorea spp.) and to generate important yield losses on this vegetative propagated crop. Within the framework of the DEVAG project (Caribbean network for the development of agroecological horticultural systems) funded by the INTERREG IV Caribbean program, the inventory, the characterization and the prevalence of viruses infecting yams in Haïti have been undertaken to assess their impact on culture and to suggest appropriate control methods. Approximately four hundred samples belonging to five yam species cultivated in Haïti (D. alata, D. bulbifera, D. cayenensis, D. rotundata, D. trifida) were collected in 2009 in farmers' fields in the five growing regions of yam. Virus detection was performed using broad spectrum PCR and RT-PCR tests targeting badnaviruses, CMV (genus Cucumovirus), potexviruses and potyviruses, after direct binding of viral particles on PCR tubes. Ninety four samples were infected by at least one of those viruses. The Badnavirus genus represents the most prevalent viral type. Hence, badnavirus RnaseH gene fragment (528 bp) was detected and amplified from most of the D. bulbifera samples (15/18) and D. trifida samples (6/6), and in half of the D. alata samples (61/132). Genetic diversity of these sequences is high (50- 97%) and these sequences belong to six out of thirteen groups of yam badnaviruses identified by Kenyon et al. (2008). Analysis of the potyvirus sequences (CP - 282 bp) obtained from ten samples revealed the existence of YMMV on D. alata (7/132) and D. trifida (1/6) samples. Similarly, two strains of a new potyvirus were found in D. trifida samples (2/6) with a nucleotide identity of 74-79% with two viral species, ALiMV and PTV. Furthermore, three sequences (245 bp) related to the RdRp gene of potexviruses, with 71-78% of nucleotide identity with PAMV and PepMV were found in two D. bulbifera and D. rotundata plants. This is the first inventory of yam viruses in Haïti which highlights a high viral prevalence rate with large amount of diversity within each virus species tested. Excepted for badnaviruses for which impact on yam production is misunderstood, the pressure of RNA viruses appears to be very low for the most common varieties, but strong for the rarer cultivars suggesting that a dramatic varietal genetic erosion is underway. (Texte intégral