Jean-Heinrich Daugrois

A putative major gene for rust resistance linked with a RFLP marker in sugarcane cultivar 'R570'

Inheritance of resistance to rust was investigated in the self progeny of the sugarcane cultivar ‘R570’ also used to build a RFLP genetic map. Resistance was evaluated through both field and controlled greenhouse trials. A clear-cut 3 (resistant) ∶ 1 (susceptible) segregation indicative of a probable dominant resistant gene was observed. This is the first documented report of a monogenic inheritance for disease resistance in sugarcane. This gene was found linked at 10 cM with an RFLP marker revealed by probe CDSR29. Other minor factors involved in the resistance were also detected.

Appearances Can Be Deceptive: Revealing a Hidden Viral Infection with Deep Sequencing in a Plant Quarantine Context

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.

Geographical Distribution of Four Sugarcane yellow leaf virus Genotypes

Specific primer pairs were designed to distinguish four genotypes (BRA for Brazil, CUB for Cuba, PER for Peru, and REU for Réunion Island) of Sugarcane yellow leaf virus (SCYLV) by reverse transcription-polymerase chain reaction (RT-PCR). A unique genome fragment was amplified from each genotype, with the exception of genotypes BRA and PER that are phylogenetically relatively close and were designated genotype BRA-PER. These RT-PCR primers were then used to identify the SCYLV genotype(s) present in 18 different sugarcane growing locations in the world, and 245 leaf samples infected by the virus were analyzed. Most samples were infected by only one of the three genotypes, but mixed infections occurred. Genotype BRA-PER was found in all sugarcane growing locations, whereas genotypes CUB and REU were each found in four geographical locations only. Genotypes BRA-PER, CUB, and REU were all three detected in locally bred sugarcane cultivars in Guadeloupe, indicating local transmission of these genotypes. In contrast, only genotypes BRA-PER and CUB were found in locally bred cultivars in Brazil, whereas genotype REU was detected in this country in cultivar R570 imported from Réunion. Similarly, genotypes BRA-PER and REU are both present in Réunion, but genotype BRA-PER has not, as of yet, spread on this island. Presence of several SCYLV genotypes in Brazil, Colombia, Guadeloupe, Mauritius, and Réunion suggests different virus introductions and/or different evolution histories of the virus after its introduction into a new environment.

Experimental assessment of the accuracy of genomic selection in sugarcane

Sugarcane cultivars are interspecific hybrids with an aneuploid, highly heterozygous polyploid genome. The complexity of the sugarcane genome is the main obstacle to the use of marker-assisted selection in sugarcane breeding. Given the promising results of recent studies of plant genomic selection, we explored the feasibility of genomic selection in this complex polyploid crop. Genetic values were predicted in two independent panels, each composed of 167 accessions representing sugarcane genetic diversity worldwide. Accessions were genotyped with 1,499 DArT markers. One panel was phenotyped in Reunion Island and the other in Guadeloupe. Ten traits concerning sugar and bagasse contents, digestibility and composition of the bagasse, plant morphology, and disease resistance were used. We used four statistical predictive models: bayesian LASSO, ridge regression, reproducing kernel Hilbert space, and partial least square regression. The accuracy of the predictions was assessed through the correlation between observed and predicted genetic values by cross validation within each panel and between the two panels. We observed equivalent accuracy among the four predictive models for a given trait, and marked differences were observed among traits. Depending on the trait concerned, within-panel cross validation yielded median correlations ranging from 0.29 to 0.62 in the Reunion Island panel and from 0.11 to 0.5 in the Guadeloupe panel. Cross validation between panels yielded correlations ranging from 0.13 for smut resistance to 0.55 for brix. This level of correlations is promising for future implementations. Our results provide the first validation of genomic selection in sugarcane.

Variation in Infection Capacity and in Virulence Exists Between Genotypes of Sugarcane yellow leaf virus

Two experiments, one in Guadeloupe and one in Réunion Island, were performed to transmit different genotypes of Sugarcane yellow leaf virus (SCYLV) to eight sugarcane cultivars differing in resistance to infection by the virus and to yellow leaf. Transmission was attempted from SCYLV-infected sugarcane plants or leaves to healthy tissue-cultured plantlets grown in vitro and with the aphid vector Melanaphis sacchari. After inoculation and elimination of insects with an insecticide, plantlets were transferred to Montpellier, France and grown in a greenhouse. Plants were tested for presence of SCYLV by tissue-blot immunoassay and reverse-transcription polymerase chain reaction after 5 to 6 months of growth. SCYLV genotypes BRA-PER, CUB, and REU were detected in 47, 62, and 39% of plants inoculated with these genotypes in Guadeloupe, respectively. SCYLV genotypes BRA-PER and REU and a mixed infection of genotypes BRA-PER and REU were detected in 56, 33, and 42% of plants inoculated with these genotypes in Réunion Island, respectively. Genotypes BRA-PER and CUB could be transmitted to all eight sugarcane cultivars, but genotype REU could never be transmitted to resistant sugarcane cvs. H78-4153 and H78-3567. SCYLV genotype REU was transmitted successfully to sugarcane cv. R570 in Guadeloupe, but not in Réunion Island. Genotypes BRA-PER and CUB induced yellow leaf symptoms in susceptible or highly susceptible sugarcane cultivars, whereas genotype REU induced very few symptoms. SCYLV was not found in several symptomatic plants, suggesting an association of disease with undetectable populations of the virus or a nonviral cause. This is the first report of variation in infection capacity and in virulence of SCYLV.

Aerial Contamination of Sugarcane in Guadeloupe by Two Strains of Xanthomonas albilineans

Two sugarcane plots were set up in Guadeloupe with disease-free tissue cultured plants in a banana growing location distant from sugarcane fields. Thirteen weeks after planting sugarcane in the field, a Xanthomonas albilineans strain belonging to serotype 3 (strain XaS3) was detected in water sampled at sunrise on the leaves in the first plot. This strain randomly invaded the sugarcane canopy. Seven weeks later, a new strain belonging to serotype 1 (strain XaS1) appeared on leaves and populations of strain XaS1 progressively increased on the leaf surface, whereas populations of strain XaS3 progressively decreased. Leaf scald symptoms were first noted 26 weeks after sugarcane planting. However, only strain XaS1 was isolated from leaves and a few sugarcane stalks showing symptoms. Both strains also colonized the second field plot, which was studied at the end of the experiment to avoid human interference of aerial contamination of sugarcane. After inoculation of three sugarcane cultivars by the decapitation technique, strain XaS1 was as virulent or more virulent than five other strains of X. albilineans isolated from diseased sugarcane plants in Guadeloupe. Although strain XaS3 colonized a few stalks, it failed to produce any symptoms and was the least virulent strain. Leaf surface colonization by X. albilineans was reproduced in a greenhouse trial by spraying the pathogen on sugarcane foliage. After 8 weeks, the pathogen was isolated from disinfected leaf blades. Although the leaf scald pathogen is thought to be mainly transmitted by infected cuttings, aerial transmission of X. albilineans is also known to occur. These results indicate the importance of sugarcane phyllosphere colonization by virulent strains in the epidemiological cycle of leaf scald disease in Guadeloupe.

High Variation in Pathogenicity of Genetically Closely Related Strains of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen, in Guadeloupe.

ABSTRACT Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.

Variation in Albicidin Biosynthesis Genes and in Pathogenicity of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

ABSTRACT Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.

Epiphytic Populations of Xanthomonas albilineans and Subsequent Sugarcane Stalk Infection Are Linked to Rainfall in Guadeloupe

Three separate field trials were established in Guadeloupe under different agronomic and rainfall conditions to study phyllosphere contamination and infection of sugarcane plants by Xanthomonas albilineans, the causal agent of sugarcane leaf scald. Disease-free and leaf scald susceptible cv. B69566 was planted and monitored during three 1-year crop cycles. Presence of leaf scald contaminated sugarcane fields in the proximity of the disease-free trials appeared critical in early contamination of the sugarcane phyllosphere. Later on, particular meteorological events, such as tropical storms, were also important in aerial spread of the pathogen. A positive correlation was found between epiphytic populations of X. albilineans and severity of leaf necrotic symptoms, but occurrence of leaf symptoms was not always related to subsequent stalk infection. However, when the data of the three crop seasons were considered together, a high correlation was found between rainfall and maximum epiphytic populations of X. albilineans, and between rainfall and subsequent stalk infections. Consequently, rainfall is a key factor to be considered in evaluation of risks of leaf scald epidemics, and protocols for propagation of healthy sugarcane material and screening methods for leaf scald resistance may have to be revised in humid tropical locations.

First Report of Sugarcane Yellow Leaf Virus in the French West Indies

Unusually severe leaf yellowing symptoms, similar to those described for yellow leaf syndrome (1), have been observed in several sugarcane clones in Guadeloupe since 1994, and since 1997 in Martinique. Leaf samples exhibiting various types of yellowing were taken from five different sugarcane clones, and analyzed by immunosorbent electron microscopy. Spherical particles, 24 to 28 nm in diameter and characteristic of luteoviruses, were found in two of five samples. The two infected samples showed yellowing on the underside of the midrib and one had a pinkish coloration on the upper side. The presence of sugarcane yellow leaf virus (ScYLV), the causal agent of sugarcane yellow leaf disease, was confirmed by reverse transcription-polymerase chain reaction (2) in these two samples and in 36 of 184 sugarcane clones bred in Guadeloupe and sent to Cirads quarantine station in Montpellier, France. Following these observations, surveys were undertaken with a tissue blot enzyme immunoassay to analyze the distribution of ScYLV in sugarcane clones in the French West Indies. The midrib base of the first visible dewlap leaf was used to detect the presence of the virus in the phloem. In a first survey, clones of various origins worldwide were taken from germplasm collections. Two to three leaf samples per clone were analyzed from 78 clones in a collection in Guadeloupe and from 36 in a collection in Mar-tinique. Fifty of the 114 clones were infected by ScYLV, and ScYLV was detected in 21 of the 32 clones exhibiting severe leaf yellowing (score 3 or higher on a 1 to 5 scale). In a second survey, 19 leaf samples were taken from each of 53 clones from plants produced by Cirads breeding program in Guadeloupe. The virus was detected in at least one sample for 25 of these 53 clones. ScYLV incidence in commercial fields was tested in Martinique in the variety B5992, which constitutes 57% of the cultivated area. Twenty leaves from different stools were sampled in six different fields, five of which had ScYLV-infected plants. The percentage of virus-infected stalks ranged from 0 to 90% whereas the percentage of stalks showing symptoms ranged from 50 to 100%. ScYLV appears widespread in the French West Indies, perhaps because a vector (Melanaphis sacchari) exists in Martinique and Guadeloupe. However, ScYLV was not found in all symptomatic plants, indicating that even if this luteovirus is a causal agent of leaf yellowing in the French West Indies, there may be other causal agents as well. References: (1) J. C. Comstock et al. Sugar J. 3:33, 1994. (2) J. C. Comstock et al. Sugar Cane 4:21, 1998.