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

Quorum sensing genes rpfF and xanB2 are not essential for albicidin production nor sugarcane colonization by Xanthomonas albilineans

Xanthomonas albilineans (Xa) produces albicidin, a unique and specific toxin that causes foliar symptoms of sugarcane leaf scald disease. In X. campestris pv. campestris, a cluster of rpf (for regulation of pathogenicity factors) genes and xanB2 are involved in control of various cellular processes. rpfF and xanB2 encode DSF (diffusible signal factor) and DF (diffusible factor), respectively, which are two quorum sensing signalling molecules. Both quorum sensing systems appear to be used by Xa, since mutation of rpfF in Florida strain XaFL07-1 resulted in reduced protease production, and mutation of xanB2 resulted in loss of xanthomonadin (yellow pigment) production. Mutations of rpfF and xanB2 were verified by PCR analyses. Mutations of rpfF and complementation in trans were also verified by use of an X. campestris DSF reporter strain. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, exhibited pencil line symptoms indicative of albicidin production on emerging leaves and colonization of leaf vessels after inoculation of stalks by the decapitation method with all mutants, including separate deletion mutations of rpfG and rpfC (encoding two sensor components of the DSF system). Preliminary experiments indicated that several rpfF and xanB2 mutants colonized sugarcane stalks as efficiently, both spatially and in intensity, as wild type Xa. Additional inoculation experiments are in progress to assess disease severity caused by rpf mutants, including deletion of the entire rpfGCF region. However, our preliminary data showed that neither DSF nor DF is essential for albicidin production or sugarcane colonization by Xa. Therefore, albicidin production and sugarcane colonization by Xa may not be controlled by quorum sensing or may involve another system. (Texte intégral

Identification of pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by Transposon Mutagenesis

Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In addition to the previously identified albicidin biosynthesis gene ciuster mutations, 33 new loci were identified in which insertions were correlated with reduced pathogenicity. These insertions affected genes predicted to encode proteins involved in a variety of functions, inciuding exopolysaccharide and lipopolysaccharide biosynthesis, fatty acid biosynthesis, regulatory and cell signaling, and secretion systems. Several of these have been associated with virulence in other bacterial plant pathogens that invade the xylem. However, sorne loci were identified that are predicted to encode previously unrecognized and apparently essential pathogenicity factors, at least for sugarcane leaf scald, inciuding an OmpA family outer membrane protein. Five independent TnS insertions in OmpA locus XALc_0557 of X albilineans strain XaFL07-1 produced no or very few leaf symptoms. These mutants produced albicidin in vitro and were able to multiply in sugarcane leaf tissue to levels similar to the wild-type strain, but did not efficiently colonize the sugarcane stalk. These ompA mutants were also affected in growth rate, motility and biofilm formation in vitro. (Résumé d'auteur

Towards identification of pathogenicity genes specific to Xanthomonas albilineans strains closely associated with sugarcane leaf disease outbreaks

Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, one of the major diseases of sugarcane. Interestingly, this pathogen lacks both the xanthan gum genes cluster and a type III secretion system (T3SS) of the Hrp1 and Hrp2 injectisome families (1). X. albilineans produces a unique and specific toxin, albicidin, which is a powerful DNA gyrase inhibitor. Consequently, albicidin blocks chloroplasts differentiation, resulting in leaf scald symptoms. High genetic and pathogenic variability exists among strains of X. albilineans, and 10 genetic groups were identified by pulsed field gel electrophoresis (PFGE). All strains involved in sugarcane leaf scald disease outbreaks since the late 1980s belong to the same genetic group called PFGE-B, whereas the strains isolated previously belong to other groups, especially to group PFGE-A. These two groups were also revealed by multilocus sequence analysis (MLSA) using seven housekeeping genes (groEL, dnaK, gyrB, atpD, efp, recA and glnA). The complete genome sequence of strain GPE PC73 belonging to PFGE-B is now available (1). To better understand the genetic differences between the PFGE-A and PFGE-B strains, Suppression Subtractive Hybridization (SSH) analysis was performed to compare the genomes of XaFL07-1 (PFGE-B) and Xa23R1 (PFGE-A), both isolated in Florida. SSH is a method used to identify DNA fragments that are uniquely found in one strain when compared with another, closely related bacterial strain (2,3). We enriched a library of unique DNA sequences from strain XaFL07-1 (tester strain), using Xa23R1 DNA as the driver strain. A total of 188 XaFL07-1-specific clones were generated and sequenced. Sequences were all compared against the GPE PC73 genome and against the GenBank non redundant database (NCBI). Initial screening focused on 12 genes with potential pathogenicity function and for which SSH data were confirmed by PCR and Southern blot hybridization. These included a DNA methyltransferase, a chemotaxis protein, a permease, a CRISPR protein, and an Rhs protein. However, after further distribution studies of these genes among strains of X. albilineans representing the genetic diversity observed in this pathogen, only one gene encoding a DNA (cytosine-5)-methyltransferase was found to be specific to PFGE-B strains. In GPE PC73, this gene is localized in a 53 kb chromosome region of phage origin that includes also two other DNA methyltransferases. SSH often results in identification of phage DNA (3); DNA methyltransferases are known to play a role in genetic regulation by modifying the binding of DNA polymerases on promoters. Additional PCR and Southern blot screening of all SSH clones, including genes with unknown functions, could also lead to the identification of other pathogenicity genes specific to PFGE-B strains of X. albilineans. Knockout mutagenesis of the DNA methyltransferases and of other SSH candidate genes will be performed to investigate the role of these genes in pathogenicity of Xanthomonas albilineans strains closely associated with sugarcane leaf scald disease outbreaks. (Texte intégral

Bioactivity-guided mass spectral net working reveals a new set of allbicidin derivatives from Xanthomonas albilineans

Albicidin is produced by the sugarcane pathogenic bacterium Xanthomonas albilineans and is a potent antibiotic specifically targeting the bacterial DNA gyrase with an IC50 value in the nanomolar range. The structure of albicidin, which has remained unsolved for more than three decades since its first description, has been solved by a combination of extensive NMR and HR-MS/MS experiments. An inherent problem for the structure elucidation was the low amount of the albicidin production in cell culture. The low production forced us to cultivate several hundred liters in order to purify a few milligrams required for NMR experiments. In comparison, LC-HR-MS/MS has the great advantage of several orders of magnitude and high sensitivity. Thus, this approach requires much less material. But more importantly, purification of compounds is not necessary for structural analysis. Due to the application of targeted LC-MS/MS experiments, we were able to propose the structure of N-terminal carba- S158 moylated and b-methoxylated albicidin derivatives. Both of them are currently chemically synthesized and studied in a medicinal chemistry campaign. In order to search for more pharmaceutically relevant albicidin derivatives, we applied non-targeted LC-HR-MS/MS approaches combined with mass spectral networking, a recently introduced approach for non-targeted MS/MS data-analysis. However, as for the structural elucidation of albicidin, the even lower concentrated derivatives resulted in difficulties to obtain useful MS/ MS spectra. Since we typically operate the mass spectrometer in data-dependent acquisition mode (DDA), it inherently selects high abundant compounds of cell extracts for MS/ MS scans and possibly ignore low abundant compounds. The same goes for the network analysis which is based on similarity of MS/MS spectra and may not take potential ion species into account. To overcome this problem, we implemented a bioactivity-guided fractionation by solid phase extraction and semi-preparative HPLC. Fractions with antibacterial activity were subsequently submitted to several LC-MS/MS runs in DDA mode with shifted survey scan windows (stitched DDA). Besides the increased amount of MS/MS events per mass range, narrowing down the m/z range of the survey scan also increased the dynamic range of the orbitrap analyzer, which enabled us to significantly increase the method's sensitivity. Finally, we were able to detect a whole series of new albicidin derivatives and to propose their structures based on exact mass determination and MS/MS fragmentation patterns. These new compounds provide great opportunities in lead optimization of albicidin as a new anti-infective drug. Apart from the pharmaceutical relevance, some of the identified by-products give us important insights into the formation of albicidin and its intriguing biosynthetic machinery. (Résumé d'auteur

Deciphering small molecules as new virulence factors in the bacterial sugarcane pathogen Xanthomonas albilineans

Xanthomonas albilineans is a xylem-invading plant pathogen that causes leaf scald disease of sugarcane. Unlike most plant pathogenic bacteria, X. albilineans does not possess a Hrp-Type Three Secretion System. Pathogenicity of this bacterial species must therefore rely on other virulence factors. X. albilineans produces albicidin, a toxin and potent inhibitor of DNA gyrase which inhibits proplastic DNA replication. Consequently, chloroplast differentiation is blocked and disease symptoms develop. Albicidin is also bactericidal at nanomolar concentrations against a range of Gram-positive and Gram-negative bacteria. This potent and novel antibiotic is especially of interest because of its activity against Escherichia coli, a species causing nosocomial diseases. Sequencing and annotation of the entire genome of X. albilineans recently revealed that X. albilineans possesses 12 large genes encoding nonribosomal peptide synthetases (NRPSs) which are located in four gene clusters covering 4% of the genome (1). One of these NRPS clusters corresponds to the previously identified albicidin biosynthesis gene cluster. The mode of action of this antibiotic was extensively studied but its structure remains unknown. Characterization of albicidin is the main bottleneck which slows development of its therapeutic application. X. albilineans is a slow growing bacterium and production yields of albicidin are low, i.e. it turned out to be extremely tedious to obtain sufficient amounts for structure elucidation. To overcome this problem, we successfully considered heterologous overproduction by transferring all albicidin biosynthesis genes into the fast growing bacterium Xanthomonas axonopodis pv. vesicatoria (2). Production of albicidin in this heterologous system already allowed us to obtain several milligrams of pure compound and promising preliminary results regarding the structural characterization of albicidin. As an example, 1H-NMR analyses showed the presence of a number of para-substituted aromates, putatively tyrosine or 4-hydroxyphenylglycines. However, the number of amide protons found in 1H-NMR spectra is lower than the number expected from the biosynthesis assembly lines, suggesting the involvement of tailoring steps during or post-NRPS biosynthesis. In silico analysis of the three other NRPS gene clusters resulted in partial prediction of the sequences of the precursor peptides synthesized by these clusters which do not resemble any peptide described to date. One of these NRPS gene clusters encodes a complete machinery predicted to be required for secretion and tailoring of small molecules: ABC transporter, MbtH like protein, isomerase, aminotransferase, acyltransferase, enoyl-CoA hydratase. The two other NRPS gene clusters encode only NRPSs which are thought to trans-act with the first one. Interestingly, the NRPSs encoded by X. albilineans share characteristics with NRPSs encoded by the root and stem-nodulating Bradyrhizobium sp. BTAi1, suggesting structural similarities between small molecules produced by these two plant interacting species. In X. albilineans, functional analyses of the phosphopantheteinyl transferase gene (which is required for activation of NRPSs) showed that these non-albicidin NRPS gene clusters are most likely involved in the biosynthesis of at least one new virulence factor. Future work will focus on characterization of the full structure of albicidin as well as the isolation and characterization of other small molecules assembled by NRPS. These compounds will be chromatographically isolated and characterized by high-resolving FTICR mass spectrometry, 2D-NMR spectroscopy and X-ray crystallography. Isolation and structural characterization of these new bioactive molecules will facilitate the annotation of biosynthesis gene clusters and the study of their role during interactions between sugarcane and X. albilineans. On a biochemical level, deciphering their role in pathogenicity should result in the identifi

CRISPR-associated sequence diversity within Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane

Co-ordinated efforts to collect and maintain cotton genetic resources have increased over the last 100 years to insure the worldwide economic value of cotton fibre and cotton by-products. The classified genetic resources of cotton are extensive and include five tetraploid species in the primary gene pool, 20 diploid species in the secondary gene pool, and 25 diploid species in the tertiary gene pool. There are at least eight major cotton collections worldwide and their status and contents are discussed. An overview of the collections suggest that there is a substantial coverage of the Gossypium genome but some recently identified species are not yet maintained and several species are underrepresented and threatened by loss of their natural habitat. Meeting the high demand for cotton genetic resources and increasing the coverage of the genus with decreasing budgets are a few of the challenges facing individual collections. These types of challenges and the opportunities for international collaboration that they create are discussed. One desirable outcome of co-ordinated efforts among collections would be finding gaps in the collections and sharing of the workload to conserve the genus. Multinational communication and collaboration are critical for the evaluation of rare and unique cotton germplasm and protection of the global cotton germplasm resources. (Résumé d'auteur

Heterologous production and characterization of albicidin, a potent DNA gyrase inhibitor

Albicidin is a toxin and an antibiotic produced by the slow-growing bacterium Xanthomonas albilineans, the causal agent of sugarcane leaf scald. Albicidin is involved in the pathogenicity of X. albilineans and inhibits the replication of chloroplastic DNA. It also inhibits DNA replication in Escherichia coli at nanomolar concentrations, whereas mammalian cells are unaffected at 8 ?g/ml. Albicidin targets DNA gyrase with features of inhibition that differ from those of other known antibiotics. It is synthesized by a unique hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway that does not resemble any other pathway described to date. The antibiotic activity of albicidin against a wide range of gram-positive and gram-negative pathogenic bacteria (Enterobacter aerogenes, E. coli, Haemophilus influenzae, Klebsiella pneumoniae, Shigella sonnei, and Staphylococcus aureus) is of interest for the development of new antibacterial drugs. Low yields of albicidin production in slow-growing X. albilineans have slowed studies of its chemical structure and potential therapeutic applications. Therefore, we have developed a heterologous system for albicidin overproduction using a Xanthomonas axonopodis pv. vesicatoria strain transformed with two plasmids harbouring the complete albicidin biosynthetic gene set. A sixty-fold increase in albicidin production was obtained when compared to albicidin production by the native host, X. albilineans. Heterologous production of albicidin was confirmed by FTICR-MS (high resolving Fournier Transform Ion Cyclotron Resonance Mass Spectrometry). (Texte intégral