Agent Of Ratoon Stunting Disease Research Articles

Screening of Sugarcane Proteins Associated with Defense against Leifsonia xyli subsp. xyli, Agent of Ratoon Stunting Disease

Sugarcane is the most important sugar crop and one of the leading energy-producing crops in the world. Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, poses a huge threat to ratoon crops, causing a significant yield loss in sugarcane. Breeding resistant varieties is considered the most effective and fundamental approach to control RSD in sugarcane. The exploration of resistance genes forms the foundation for breeding resistant varieties through molecular technology. The pglA gene is a pathogenicity gene in L. xyli subsp. xyli, encoding an endopolygalacturonase. In this study, the pglA gene from L. xyli subsp. xyli and related microorganisms was analyzed. Then, a non-toxic, non-autoactivating pglA bait was successfully expressed in yeast cells. Simultaneously the yeast two-hybrid library was generated using RNA from the L. xyli subsp. xyli-infected sugarcane. Screening the library with the pglA bait uncovered proteins that interacted with pglA, primarily associated with ABA pathways and the plant immune system, suggesting that sugarcane employs these pathways to respond to L. xyli subsp. xyli, triggering pathogenicity or resistance. The expression of genes encoding these proteins was also investigated in L. xyli subsp. xyli-infected sugarcane, suggesting multiple layers of regulatory mechanisms in the interaction between sugarcane and L. xyli subsp. xyli. This work promotes the understanding of plant–pathogen interaction and provides target proteins/genes for molecular breeding to improve sugarcane resistance to L. xyli subsp. xyli.

Early Detection of Ratoon Stunting Diseases in Sugarcane Seeds Using Polymerase Chain Reaction Method

Sugarcane is a kind of plantation crop that can be used as a raw material for the sugar industry. The productivity of sugarcane can be influenced by several factors, including infection of pathogenic bacteria such as Leifsonia xyli f.sp. xyli (Lxx), the causal agent of Ratoon Stunting Disease (RSD) in sugarcane. The disease is one of the major diseases of sugarcane worldwide. RSD is symptomless unless the disease incidence is severe. The Polymerase Chain Reaction (PCR) method is generally known for detecting plant pathogens. However, there is still limited information on the detection of RSD using PCR. Therefore, our study aimed to detect the existence of RSD in sugarcane seeds using the PCR method. Three sugarcane varieties were used in this study, including AAS Agribun, AMS Agribun, and BQ (positive control of RSD). We did sample the stems and midribs of those three varieties at the age of 3 and 7 months. The results showed that Lxx was found both in the stem and midrib of sugarcane variety BQ at the age of 3 and 7 months. These results indicated that the PCR method can be used for early detection of RSD in sugarcane seeds.

Kasugamycin on Leifsonia xyli subsp. xyli in the in vitro culture of sugarcane

ABSTRACT: One of the significant obstacles to the growth of sugarcane production is the infection by phytopathogens, mainly by the bacterium Leifsonia xyli subsp. xyli (Lxx) causal agent of Ratoon stunting disease. Thus, this research aimed to evaluate the effects of kasugamycin on the in vitro growth of sugarcane, as well as its effect on the bacterium Lxx. Explants of strain SP791011 from sugarcane were inoculated in MS culture medium supplemented with the antimicrobial kasugamycin at concentrations of 0.00; 0.87; 1.08; 1.74 and 3.48 mL.L-1, where they remained for 30 days. After this period, the survival rate, shoot number per explant, height of the explants, phytomass, dry phytomass and phytosanitary were evaluated based on the presence of genomic DNA of Lxx. It was verified that the culture in kasugamycin influenced the morphological variables negatively; nevertheless, the antimicrobial did not demonstrate phytotoxicity to the plants. All treatments tested in this experiment were diagnosed as positive, with DNA amplification for Lxx, despite it was observed a reduction in bacterial load, suggesting that kasugamycin at higher doses can be evaluated as an attempt to eliminate the bacterium in the in vitro cultivation of sugarcane.

Molecular Detection of Diverse Leifsonia Strains Associated With Sugarcane.

Leifsonia xyli subsp. xyli, causal agent of ratoon stunting disease (RSD) of sugarcane (Saccharum interspecific hybrids), is the most well-known member of the Microbacteriaceae genus Leifsonia. However, the presence of other Leifsonia strains associated with sugarcane has not been reported. A total of 697 Australian and 40 Indonesian sugarcane fields were screened by leaf sheath biopsy (LSB) PCR using primers specific for L. xyli subsp. xyli, in addition to primers designed to amplify DNA from other members of the genus Leifsonia. While L. xyli subsp. xyli was detected in 126 fields, a total of 37 distinct and novel Leifsonia and non-Leifsonia strains were detected in 116 fields. Representatives of these strains were also detected in multiple samples of expressed xylem sap. Sequencing and phylogenetic analyses demonstrated the presence of a broad complex of novel Leifsonia strains, in addition to strains closely related to the recently erected Cnuibacter genus. Attempts to isolate Leifsonia strains were unsuccessful; however, one strain related to Cnuibacter was recovered from expressed xylem sap. Among the genetically diverse lineages discovered, identical genotypes were present in multiple sugarcane varieties growing in disparate regions in different years, strongly suggesting an ongoing association with sugarcane. The epidemiological significance of these strains is unknown, but there is evidence that they can interfere with serological and microscopic RSD diagnostics, and there is the potential that they may represent new and distinct pathologies of sugarcane.

Status of ratoon stunting disease of sugarcane (Leifsonia xyli subsp. xyli) in Nigeria

Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane (RSD), is a major constraint to sugarcane production worldwide. In Nigeria, the presence of RSD has not been conclusively determined. This study investigated the presence or absence of RSD through surveys in 18 sugarcane growing states in Nigeria. Sugarcane plants or ratoon crops of 7–12 months of age were randomly sampled, and a total of 1190 samples of sugarcane stem cuttings from 76 cultivars were collected. The lowest four internodes were cut longitudinally into nodes, and examined internally for reddish comma-like discoloration. PCR was conducted on DNA extracted from sugarcane sap using Lxx-specific primers. Reddish comma-like internal symptoms of RSD were observed only in samples of cultivar Co510. None of the sugarcane samples, including those from Co510, yielded the 438 bp band expected for PCR detection of Lxx. Immunofluorescence microscopy analyses performed at SASRI, South Africa, using sap from the collected sugarcane samples, also did not detect the bacterium. Thus, the presence of RSD of sugarcane in Nigeria was not confirmed, contrary to its reported presence in 1956. Strict quarantine procedures should be followed to prevent introduction of the pathogen into Nigeria.

COMPARISON OF CONVENTIONAL, NESTED AND REAL-TIME PCR FOR DETECTION OF THE CAUSAL AGENT OF RATOON STUNT IN IRAN

Leifsonia (Clavibacter) xyli subsp. xyli (Lxx) is the etiological agent of ratoon stunting disease (RSD), one of the most important diseases affecting productivity of sugarcane crops. The lack of defined external symptoms in the plant is a challenge for the diagnosis of RSD. We compared sensitivity of three methods including conventional PCR, nested PCR and real- time PCR for detection of Lxx sugarcane fields of the Khuzestan province (Iran). Total genomic DNA of infected sugarcanes was extracted and various amounts of it used as template for three mentioned techniques. The results showed that conventional PCR lacks sufficient sensitivity to be used as a diagnostic tool for RSD. Nested PCR had acceptable sensitivity, however, this technique is time-consuming. Real-time PCR is the most efficient method but it is expensive and requires specialist for analyzing the data. Totally, each of these methods has its own advantages and disadvantages. Therefore, the election of these techniques for diagnosis depends on factors like disease severity, the goal and available facilities.

Quick detection of Leifsonia xyli subsp. xyli by PCR and nucleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates

A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in xylem sap samples from stalks. After removal of abiotic impurities and large molecular weight microorganisms by a 3000 rpm, 5 min centrifugation, the Lxx bacteria were precipitated from the xylem sap by a 12,000 rpm, 10 min centrifugation. The Lxx cells were re-suspended in 50 μl freshly prepared Buffer A (0.1M NaOH + 2% Tween 20), lysed by heating at 95°C for 10 min, cooled down on ice for 3 min, and neutralized by mixing with 50 μl of freshly prepared Buffer B (0.1M Tris-HCl, pH 8.0 + 2 mM EDTA). The resulting lysates were directly subjected to conventional PCR with Lxx-specific primers. Of the 31 varieties tested, 19 were positive for Lxx infection, including all FN, GT, and YT varieties. These varieties also tested positive by PCR on DNA templates extracted from xylem sap samples using a CTAB procedure and by two enzyme-immunoassays, dot-blot (DBEIA) and direct antigen coating (DAC-ELISA). DB-EIA and DAC-ELISA detected Lxx in 90.3 and 93.5%, respectively, of the varieties while the detection rate with PCR was 61.3%. The modified PCR assay was quick and more economical. It did not require organic solvent usage or ethanol precipitation, but produced the same level of detection as that of the PCR using DNA samples prepared by the CTAB procedure. All the PCR amplicons were 439 bp in size sharing the same nucleotide sequence. This consensus sequence, GenBank Accession EU723209, aligned perfectly with three other Lxx nucleotide sequences available in the GenBank, AE016822, AF034641, and DQ232616. However, two mis-matches, a (G?A) transversion and a deletion, were found in another nucleotide sequence AF056003.

Characterization of new IS elements and studies of their dispersion in two subspecies of Leifsonia xyli

BackgroundLeifsonia xyli is a xylem-inhabiting bacterial species comprised of two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). Lxx is the causal agent of ratoon stunting disease in sugarcane commercial fields and Lxc colonizes the xylem of several grasses causing either mild or no symptoms of disease. The completely sequenced genome of Lxx provided insights into its biology and pathogenicity. Since IS elements are largely reported as an important source of bacterial genome diversification and nothing is known about their role in chromosome architecture of L. xyli, a comparative analysis of Lxc and Lxx elements was performed.ResultsSample sequencing of Lxc genome and comparative analysis with Lxx complete DNA sequence revealed a variable number of IS transposable elements acting upon genomic diversity. A detailed characterization of Lxc IS elements and a comparative review with IS elements of Lxx are presented. Each genome showed a unique set of elements although related to same IS families when considering features such as similarity among transposases, inverted and direct repeats, and element size. Most of the Lxc and Lxx IS families assigned were reported to maintain transposition at low levels using translation regulatory mechanisms, consistent with our in silico analysis. Some of the IS elements were found associated with rearrangements and specific regions of each genome. Differences were also found in the effect of IS elements upon insertion, although none of the elements were preferentially associated with gene disruption. A survey of transposases among genomes of Actinobacteria showed no correlation between phylogenetic relatedness and distribution of IS families. By using Southern hybridization, we suggested that diversification of Lxc isolates is also mediated by insertion sequences in probably recent events.ConclusionCollectively our data indicate that transposable elements are involved in genome diversification of Lxc and Lxx. The IS elements were probably acquired after the divergence of the two subspecies and are associated with genome organization and gene contents. In addition to enhancing understanding of IS element dynamics in general, these data will contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxc and Lxx.

Putative lipoproteins identified by bioinformatic genome analysis of Leifsonia xyli ssp. xyli, the causative agent of sugarcane ratoon stunting disease

SUMMARY Leifsonia xyli ssp. xyli is the causative agent of ratoon stunting disease, a major cause of economic loss in sugarcane crops. Understanding of the biology of this pathogen has been hampered by its fastidious growth characteristics in vitro. However, the recent release of a genome sequence for this organism has allowed significant novel insights. Further to this, we have performed a bioinformatic analysis of the lipoproteins encoded in the L. xyli genome. These analyses suggest that lipoproteins represent c. 2.0% of the L. xyli predicted proteome. Functional analyses suggest that lipoproteins make an important contribution to the physiology of the pathogen and may influence its ability to cause disease in planta.

Establishment of a Functional Genomics Platform for Leifsonia xyli subsp. xyli

Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

A method for rating sugarcane cultivars for resistance to ratoon stunting disease based on an enzyme-linked immunoassay

The evaporative-binding enzyme-linked immunosorbent assay (EB-EIA) was used to compare the relative populations of Leifsonia (Clavibacter) xyli subsp. xyli, the causal agent of ratoon stunting disease, in 25 cultivars of sugarcane that were inoculated by planting them through a contaminated mechanical planter. There were highly significant differences between cultivars in relative populations of bacteria in xylem extracts. However, the percentage of samples rated as positive for the bacterium was consistently lower in only one cultivar, H56–752. The absorbance readings for cultivars between the plant and ratoon crop and between two separate experiments were highly correlated. The EB-EIA is a simple method for rating cultivars for resistance to ratoon stunting disease.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction‐based assay

A sensitive, specific polymerase chain reaction‐based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

First Report of Leaf Scald Disease and Ratoon Stunting Disease of Sugarcane in French Guyana.

In December 1995, leaf scald symptoms were observed in sugarcane (Saccharum sp.) cultivar B64277 in French Guyana. Symptomatic plants occurred both in a sugarcane germplasm collection near the road between Sinnamary and Saint-Elie and in a nursery near Sinnamary. Sugarcane imported from Martinique had been used to establish the germplasm collection that in turn had been used to establish the nursery. Ten-month-old mature plants in the germplasm collection had abnormal side shoots on the lower part of the stalks and suckers (nonmillable stalks) with white scalded areas on leaves. Leaves on 1-month-old shoots in the nursery exhibited chlorosis and white, pencil-line streaks. Samples prepared from symptomatic stalks from the two locations were plated on a selective medium (1), and two isolates of Xanthomonas albilineans were recovered. Both of these isolates caused leaf scald symptoms on leaves of sugarcane cultivar B69566 inoculated by a decapitation technique, and belong to serovar 3 previously reported in the Caribbean from Guadeloupe, Martinique, and St. Kitts. The RFLP (restriction fragment length polymorphism) pattern of these two isolates was different from the 54 patterns among 218 other strains collected throughout the world (2), but similar to the pattern of a strain of serovar 3 from Martinique. This indicated that the pathogen might have been introduced with cuttings imported from Martinique. Three stalks of mature cane from varieties B5992, B64277, and R570 from the germplasm collection were tested for the presence of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease. Immunofluorescence tests on sap (3) revealed the presence of the pathogen in the three stalks of B64277. All sugarcane plants in the nursery and the germplasm collection were destroyed by the use of glyphosate sprays in January 1996 in an attempt to arrest the spread of the two bacterial pathogens. In order to obtain healthy seed cane for future planting, a new germplasm collection of 0.6 ha and consisting of 11 cultivars was planted in January 1996 with disease-free, tissue-cultured plants provided by the CIRAD sugarcane breeding station in Guadeloupe.

Título en español.

Este estudio presenta los resultados de experimentos llevados a cabo en el laboratorio con el objeto de determinar si el agente causal del enanismo del retoño de la caña de azúcar puede trasmitirse a través del suelo. Los resultados demuestran que no ocurrió tal trasmisión, aún cuando las plantas se desarrollaron en el suelo en estrecho contacto unas con otras. Tampoco se observó caso alguno de trasmisión en aquellas plantas que, aunque sembradas en distintos tiestos, se mantuvieron lo suficientemente juntas como para que sus hojas se entrelazaran durante el curso del experimento. Por lo tanto, los resultados obtenidos indican que la infección, o bien la reinfección de la semilla tratada con calor para inactivar el virus es sumamente difícil, si no imposible a través del suelo.