Jean-Claude Girard

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.

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.

Impact of Sugarcane yellow leaf virus on Sugarcane Yield and Juice Quality in Réunion Island

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Réunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Réunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.

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.

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.

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 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 Acidovorax avenae subsp. avenae causing sugarcane red stripe in Gabon.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 1- to 3-mm wide and several dm long dark red stripes were observed on sugarcane (Saccharum spp.) leaves of many plants of cultivar R570. These plants were 5.5 months old in the first ratoon crop. Additionally, spindle leaves of several stalks were rotted and could be easily pulled out of the top of the stalk. Longitudinal sections of diseased stalks showed reddish-brown discoloration of the upper stem and the rotted spindle had an unpleasant odor. Circular, convex, smooth, yellow-cream pigmented bacterial colonies with 2 to 3 mm diameter were isolated after 3 days at 28°C from young leaf lesions on YPGA (yeast extract 7 g/L, peptone 7 g/L, glucose 7 g/L, agar 15 g/L, pH 6.8 to 7.0). The 16S-23S internal transcribed spacer (ITS) of two representative colonies was PCR amplified, and the nucleotide sequences were shown to be 99% identical to the 16S-23S ITS sequence from the genome of Acidovorax avenae subsp. avenae strain ATCC 19860 (GenBank: CP002521.1). One of these A. avenae subsp. avenae isolates from Gabon was inoculated into greenhouse grown plants of sugarcane cultivar R570. Plants were inoculated by injection into the sheath of spindle leaves above the meristem with the bacterial strain (12 plants) or with a water control (six plants). In this method, the bacteria (108 CFU/ml) were injected using a syringe through the leaf sheath until filling the leaf whorl. Three weeks post-inoculation, one to several cm long red-brown stripes were observed on leaves of 11 of 12 inoculated plants. Seven weeks post-inoculation, all plants exhibited symptoms, from red, brown, or black stripes to leaf necrosis, rotting, and death of the spindle leaves (six plants). All six control plants were symptomless. In a second experiment, 6 of 12 plants showed symptoms 3 weeks post inoculation, and the pathogen was successfully re-isolated from all six symptomatic plants with YPGA medium. The 16S-23S ITS of three single colonies obtained each from different symptomatic plants were PCR amplified and the nucleotide sequences were again found 99% identical to the 16S-23S ITS sequence from the genome of A. avenae subsp. avenae ATCC 19860. To our knowledge, this is the first report of A. avenae subsp. avenae, the causal agent of sugarcane red stripe (also reported as top rot), in Gabon. It is also the first description of the occurrence of the top rot form of the disease in R570, a cultivar that is grown in several locations of Africa, the Mascarene Islands, and the French West Indies. A large-scale survey needs to be undertaken to determine the distribution of red stripe in Gabon, a disease for which several outbreaks have been reported recently worldwide (1,2). References: (1) M. P. Grisham and R. M. Johnson. Phytopathology 101:564, 2011. (2) S. Zia-ul-Hussnain et al. Afr. J. Biotechnol. 10:7191, 2011.

First report of #Leifsonia xyli# subsp. #xyli#, causal agent of ratoon stunting of sugarcane, in Jamaica

To our knowledge, this is the first report that Leifsonia xyli subsp. xyli, previously named Clavibacter xyli subsp. xyli (2), has been detected and identified in sugarcane in Jamaica. Although ratoon stunting (also known as ratoon stunting disease or RSD) has been reported in Jamaica since 1961, presence of the pathogen had never been confirmed in symptomatic tissues. A major industry-wide survey conducted in 1987 using the fluorescent antibody staining technique failed to detect positives in any of the 61 fields sampled in Jamaica. A new survey was conducted in 2004 on eight estates and the Sugar Industry Research Institute (SIRI) in Jamaica. Six arbitrarily selected stalks were sampled from each of 64 fields representing 25 different sugarcane cultivars. A 1-cm diameter core was extracted from the center of the bottom part of the stalk and used to detect the pathogen by tissue blot immunoassay (TBIA) (3). L. xyli subsp. xyli was detected in 26 of 384 samples (7%). At least one positive sample was found in 10 fields and seven cultivars and in one case (sugarcane cv. D14146 at the St Thomas Sugar Estate), all six stalks sampled in a field were positive. The highest number of infected fields (6 of 10) occurred at Worthy Park where cane yield in 2004 was 86.54 tons per ha compared with an average of 68.04 tons per ha for major estates in Jamaica (1). This latter result would indicate that where good quality agronomic practices are maintained, the effect of ratoon stunting might not be substantial or that sugarcane cultivars grown at this location were resistant to ratoon stunting. Pathogen identification was confirmed using nested polymerase chain reaction (PCR) with three samples from a TBIA-positive field of cv. D14146. Primary primers were RSD 33 (CTGGCACCCTGTGTTGTTTTC) and RSD 297 (TTCGGTTCTCATCTCAGCGTC) and secondary, nested primers were RST60 (TCAACGCAGAGATTGTCCAG) and RST59 (CGTCTTGAAGACACAGCGATGAG). The thermocycler parameters were denaturization at 94°C for 4 min, 31 cycles at 94°C for 30 s, 55°C for 30 s, 65°C for 1 min, and final extension at 65°C for 3 min. The nested-PCR product (approximately 230 bp) of each sample was cloned and sequenced. It showed 99 to 100% identity with the 16S-23S intergenic spacer region of L. xyli subsp. xyli, thus confirming occurrence of ratoon stunting in Jamaica. Since this study, the SIRI has installed a hot-water treatment plant and will heat-treat cuttings before planting the nurseries with new sugarcane clones selected for release to growers. The SIRI will also conduct screening for ratoon stunting resistance to ensure that susceptible clones are not released to the industry. Meanwhile, the SIRI will do a more intense survey so that a more comprehensive picture may be obtained of the presence of ratoon stunting in Jamaica. References: (1) Anonymous. Annual Report of the Sugar Industry Research Institute, Jamaica, 2004. (2) L. I. Evtushenko et al. Int. J. Syst. Evol. Microbiol. 50:371, 2000. (3) N. A. Harrison and M. J. Davis. Phytopathology 78:722, 1988.

First Report of Sugarcane Leaf Scald in Gabon Caused by a Highly Virulent and Aggressive Strain of Xanthomonas albilineans

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 0.5 to 1 cm wide chlorotic stripes covered with many small red streaks were observed on sugarcane (Saccharum spp.) leaves of a single plant of cultivar R581. After removal of the leaves covering the base of the stalks, abnormal development of basal side shoots was also observed. Transversal sections of a diseased stalk showed reddening of the vessels near the nodes. Circular, convex, smooth, shiny, translucent, non-mucoid, honey-yellow pigmented bacterial colonies were isolated from stalk pieces and side shoots on XAS selective agar medium (1). The nucleotide sequence of the 16S-23S internal transcribed spacer (ITS) of a representative colony was shown to be 100% identical to the 16S-23S ITS sequence from the genome of Xanthomonas albilineans strain GPE PC73 (GenBank: FP565176.1). This strain from Gabon was named GAB266. Sugarcane stalks of greenhouse grown cultivar CP68-1026 were inoculated with X. albilineans strains XaFL07-1 from Florida, GPE PC73 from Guadeloupe, and GAB266. Five stalks were inoculated by the modified decapitation method (3) with each strain or with a water control. One month post-inoculation (MPI), white pencil lines and severe necrosis were observed on leaves inoculated with strains XaFL07-1 and GPE PC73, and no disease symptoms appeared on non-inoculated leaves that developed 2 to 3 MPI. These results are in agreement with those generally obtained after inoculation of susceptible sugarcane cultivars with X. albilineans strains from various geographical locations under greenhouse conditions (Rott, unpublished results). In contrast, 1 MPI, only discrete white to red pencil lines were observed along with necrosis on leaves inoculated with strain GAB266, and by 2 to 3 MPI, all five inoculated plants were wilted. The pathogen was successfully reisolated by the stalk blot inoculation technique (3) with XAS medium, from all five inoculated stalks and from 98 of 114 internodes. In a second greenhouse experiment, the same three strains of X. albilineans were inoculated as described above into five sugarcane cultivars differing in resistance to leaf scald in Guadeloupe (2) (CP68-1026, highly susceptible; B69566, susceptible; R570, tolerant; B8008, resistant; Co6415, highly resistant). The same symptoms as those described above were again observed on inoculated leaves of the five sugarcane cultivars 1 MPI. Strains XaFL07-1 and GPE PC73 produced occasionally a single pencil line on non-inoculated leaves 2 to 3 MPI, but only strain GAB266 caused leaf scalding and/or plant death 2 to 3 MPI: cultivar CP68-1026 (5 of 5 plants), B69566 (5 of 5 plants), R570 (4 of 5 plants), B8008 (5 of 5 plants), and only non-inoculated leaves of cultivar Co6415 remained symptomless (5 plants). Strain GAB266 from Gabon appeared, therefore, more virulent and aggressive than the two strains of X. albilineans from Florida and Guadeloupe. To our knowledge, this is the first report of leaf scald of sugarcane in Gabon and the first description of an unusual highly virulent and aggressive strain of X. albilineans. A large-scale survey needs to be undertaken to determine the distribution of leaf scald disease and this new pathotype/race of X. albilineans in Gabon and other geographical locations. References: (1) M. J. Davis et al. Plant Dis. 78:78, 1994. (2) P. Rott et al. Phytopathology 87:1202, 1997 (3) P. Rott et al. Mol. Plant-Microbe Interact. 24:594, 2011.