Michèle Chatenet

Genetic diversity in the coat protein coding region of eighty-six sugarcane mosaic virus isolates from eight countries, particularly from Cameroon and Congo

Summary. Fifty-eight sugarcane virus isolates were obtained from leaves showing mosaic symptoms, and collected in Cameroon (26 isolates), Congo (20 isolates), Egypt (1 isolate), South Africa (3 isolates) and the U.S.A. (8 isolates). All these isolates belonged to Sugarcane mosaic virus (SCMV) based on the amplification product obtained by RT-PCR with SCMV-specific primers. The amplicons (0.9 kb) from the coat protein (CP) coding region were cloned, sequenced and compared to each other as well as to the sequences (GenBank accessions) of 16 SCMV isolates from sugarcane (Australia, South Africa and U.S.A.) and 12 SCMV isolates from maize (Australia, Germany and China). Maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the SCE or sugarcane group that included all isolates from sugarcane and the MZ or maize group that contained all isolates from maize. The 86 virus isolates were distributed in 13 minor phylogenetic groups, four (I–IV) restricted to maize and nine (V–XIII) to sugarcane. A strong correlation was observed between the sugarcane groups and the geographical origin of the SCMV isolates. Each SCMV type strain from sugarcane (A, B, D, E and SC) was distributed in a different phylogenetic group or subgroup. The 26 isolates from Cameroon constituted a relatively homogenous group (group V) whereas the 20 isolates from Congo belonged to two other groups (VI and VII). All the isolates from Cameroon and Congo were different from the SCMV type strains and other strains or isolates studied so far. It appears, therefore, that the population of SCMV from sugarcane in Africa contains virus genotypes that have not yet been described.

Detection of Sugarcane yellow leaf virus in Quarantine and Production of Virus-free Sugarcane by Apical Meristem Culture

Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRADs sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.

Intraspecific genomic variation within Xanthomonas albilineans, the sugarcane leaf scald pathogen.

ABSTRACT To better understand the nature of recent outbreaks of leaf scald disease of sugarcane in a number of sugarcane production regions of the world including Florida, Guadeloupe, Louisiana, Mauritius, Taiwan, and Texas, a study of the worldwide genetic variation of the pathogen was undertaken. A total of 218 strains from 31 geographic locations were examined. Genomic DNA of each strain was digested with the rare cutting restriction enzyme SpeI, and the fragments were separated by pulsed-field gel electrophoresis (PFGE). A total of 102 bands were identified, and 54 different DNA banding patterns (haplotypes) were observed. Eight groups of banding patterns, designated PFGE groups A through H, were consistently detected by visual, principal component, and cluster analyses. Five groups were comprised of multiple haplotypes representing numerous strains, and three were comprised of single haplotypes representing one strain each. The leaf scald outbreaks in Florida, Louisiana, Texas, and possibly Guadeloupe and Taiwan could be attributed to the introduction of strains belonging to PFGE group B. When infection by two strains each of the newly introduced strains (PFGE group B) and those previously present in Florida (PFGE group A) was analyzed in 22 sugarcane cultivars by reisolation 24 weeks after inoculation, a significantly greater mean frequency was detected for PFGE group B strains and no cultivar by PFGE group interaction was observed. Inadvertent dispersal of the pathogen among plants, possibly by means of aerosols or splashing water, was detected in a subsequent experiment. Strains of PFGE group B were recovered from the internal tissues of some plants inoculated with PFGE group A strains and were also found to be epiphytic colonizers of nonsymptomatic, noninoculated plants adjacent to the inoculated plants; whereas strains of PFGE group A were recovered only from plants that had been inoculated with them. Thus, the possibility became more apparent that strain variation might be associated, at least in part, with factors governing plant-to-plant spread of the pathogen in nature.

Distribution of sugarcane mosaic and sugarcane streak mosaic virus in India

Seven (sugarcane and sorghum) leaf samples exhibiting mosaic symptoms and that originated from (Uttar Pradesh, Maharashtra and Tamil Nadu) were characterized through RT-PCR assays with specific designed primers forSugarcane mosaic virus, SCMV (F4 and R3),Sugarcane streak mosaic virus, SCSMV(ST2 and P1) andSorghum mosaic virus, SrMV (F3 and R3). SCMV was detected by RT-PCR in 3 sugarcane varieties. The expected 0.9 kbp SCMV fragment was amplified by RT-PCR with the SCMV F4 and SCMV R3 primers with mosaic samples from Maharashtra (cv.VSI-9-20 and CoM 9086) and Tamil Nadu (cv. Co740). However, ca 0.5 kbp fragment was amplified from six of the samples out of seven tested using primers SCSMV-ST2 and SCSMV-P1. RT-PCR reactions were negative with SrMV primers pair in all the tested samples. Our results suggested that SCSMV is found more commonly associated with sugarcane mosaic samples in India in comparison to the SCMV. Interestingly, mixed infection of SCMV and SCSMV was also detected in sugarcane mosaic infected leaf samples from Maharashtra and Tamil Nadu.

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.

First report of sugarcane yellow leaf virus in Peru.

Two sugarcane cultivars, H 50-7209 and H 32-8560, have exhibited unusual, severe leaf yellowing for more than 18 years at Agro Industrial Paramonga S.A. (AIPSA) in Peru. In 1999, these varieties occupied about 4,600 ha (74% of the cultivated area), and almost all fields showed these symptoms. Symptoms first appear on the upper third of the leaf blades, which turns light green to light yellow in young canes up to 6 to 8 months of age. Between 10 and 16 months of age, the symptoms are visible on the spindle and first to third visible dewlap leaves. Tips and margins of older leaves become necrotic, and leaves can turn completely necrotic as the necrosis progresses down the leaves. The abaxial surface of leaf midribs is rarely bright yellow, which differs from the characteristic symptom of yellow leaf syndrome caused by the Sugarcane yellow leaf virus (ScYLV) (1). The most severe symptoms occur when the leaves of stalks that flower turn completely yellow and die. Samples from 98 plants exhibiting different types of yellowing were collected from six commercial fields of cultivars H 50-7209 and H 32-8560 and the germ plasm collection (cultivars PCG 59-1609, Trojan, CP 48-103, CP 72-2086, Q 87, and PR 908) at Paramonga. Tissue blot immunoassay was used to detect ScYLV in the midrib of the top visible dewlap leaf using antiserum provided by B. E. L. Lockhart (University of Minnesota) (2). ScYLV was detected in all 49 commercial field samples and in 35 out of 49 germ plasm samples. All six cultivars of the germ plasm collection were found to be infected, but ScYLV was detected in only a few leaves of Trojan and CP 72-2086. Eighteen cuttings from diseased stalks of cultivars H 50-7209 and H 32-8560 were grown in a greenhouse in Montpellier, France. Yellowing of the underside of the midribs and of the leaf tips appeared after 3 months in cultivar H 50-7209 but only after 9 months in cultivar H 32-8560. At 9 months, the top leaf with a visible dewlap and the four leaves immediately below it of cultivar H 50-7209 exhibited severe yellowing. Reverse transcription polymerase chain reaction with specific ScYLV primers, provided by M. S. Irey (U.S. Sugar Corp., Clewiston, FL) were used to detect ScYLV in the top visible dewlap leaf (1), and ScYLV was found in all nine samples taken from 6-month-old plants of the two cultivars. This is the first report of ScYLV in Peru. References: (1) J. C. Comstock et al. Sugar Cane 4:21, 1998. (2) S. Schenck et al. Sugar Cane 4:5, 1997.

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. References: (1) M. J. Davis et al. Plant Dis. 78:78, 1994. (2) M. J. Davis et al. Phytopathology 87:316, 1997. (3) M. J. Davis and J. L. Dean. Plant Dis. 68:896, 1984.