First Report of Xanthomonas hortorum pv. carotae Causing Bacterial Leaf Blight of Carrot in Mauritius.

Bacterial blight of carrot (Daucus carota) is caused by Xanthomonas hortorum pv. carotae (4). The pathogen is seed transmitted and carrot seeds can be an important source of primary inoculum (2). A 2008-2009 outbreak of a disease resembling bacterial blight was observed in Mauritius in 10 ha of carrot crops, primarily in humid areas of the island, at an estimated incidence of 10%. Carrot leaves with angular, water-soaked leaf spots that turned necrotic were collected at Plaine Sophie, Mauritius in December 2008. Yellow, Xanthomonas-like colonies were isolated onto KC agar medium (3). MultiLocus sequence analysis (MLSA) with four genes (atpD, dnaK, efp, and gyrB) was performed as described previously (1) on five carrot strains together with two reference strains of X. hortorum pv. carotae (LMG 8643 and LMG 8644). The reference strains were identical. Of the five Mauritius strains, two (LG1-1 and LG1-4) were identical, and most closely related to, but distinct from, the reference strains (genetic distance of 0.02). The other three strains represented two sequence types identified as Xanthomonas sp. based on a phylogenetic tree derived from concatenated sequences, but were not related to any type strain. PCR assays with a 3S primer pair specific for X. hortorum pv. carotae (2) produced an amplicon of approximately 350 bp from isolates LG1-1, LG1-4, and each of the reference strains. A PCR assay with a 9B primer pair (2) yielded an amplicon of 0.9 kb for strains LG1-1, LG1-4, and LMG 8644, whereas LMG 8643 yielded an amplicon of approximately 2.0 kb (2). Foliage of 4-week-old plants (36 plants per strain) of the carrot cv. Senator F1 were spray inoculated with a suspension of each strain using an 18-h culture in sterile 0.01 M tris buffer (pH 7.2) with approximately 1 × 108 CFU/ml. Plants sprayed with tris buffer were used as a negative control treatment. Plants were incubated in a growth chamber at 26 ± 1°C at a relative humidity of 95 ± 5% and a photoperiod of 16 h. Water-soaked lesions that developed into necrotic areas were observed 12 to 15 days after inoculation of LG1-1, LG1-4, and the two reference strains. Bacteria were recovered from lesions onto KC medium (3) 3 weeks after inoculation with mean Xanthomonas populations of at least 1 × 107 CFU/lesion. Colonies with morphology typical of Xanthomonas were recovered and typed using atpD sequencing to fulfill Koch's postulates. Although Xanthomonas-like bacteria were isolated from symptomatic carrot leaves in Mauritius in 1989, the results of that study were not published. To our knowledge, this is the first report of molecular and pathological characterization of this pathogen in carrot crops in Mauritius. References: (1) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (2) X. Q. Meng et al. Plant Dis. 88:1226, 2004. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

First Report of Tomato yellow leaf curl virus in Tomato in Mauritius.

Tomato is a food crop of economic importance in Mauritius. It is grown in open fields and in greenhouses by more than 4,500 small- and large-scale growers throughout the island. Open-field tomatoes are mostly a cooking type, while those produced in greenhouses are salad types. Acreage under production is approximately 900 ha with an annual production of approximately 11,500 tons. In September 2009, plants with reduced leaf size, leaf curling, and yellow margins associated with plant dwarfism were observed in open-field tomato crops in the southern part of the island. Whitefly populations were observed in these fields. These symptoms were suggestive of infection with a leaf curl-causing begomovirus such as Tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus). Similar symptoms caused by TYLCV were reported in neighboring Reunion Island in 1997 (1). In October 2009, 3.15 ha of tomato were surveyed in the south at la Flora, Camp diable, L'escalier, Plein Bois, and Plaine Magnien to monitor the disease. Symptomatic plants were observed in all areas surveyed and disease incidence ranged from 5 to 50%. The disease was more prevalent in tomato 'Swaraksha' and 'Epoch', which are widely cultivated. Seventeen symptomatic leaf samples from La flora, Camp Diable, L'escalier, Plein Bois, and Plaine Magnien areas were collected for begomovirus detection by PCR. Total DNA was extracted and tested using AV494 (5'-GCC YAT RTA YAG RAA GCC MAG-3') and AC1048 (5'-GGR TTD GAR GCA TGH GTA CAT G-3') primers from the core region of the coat protein that detect most begomoviruses (2). Seventeen of 17 samples (100%) gave an amplicon of expected size. PCR amplicons from selected samples were cloned and sequenced. The consensus sequence was assembled, and the sequence (GenBank Accession no. HM448447) had 100% identity with nucleotides 458 to 1,036 of the Almeria isolate (GenBank Accession no. AJ489258), an isolate from the Netherlands (FJ439569), Morocco (EF060196), and Spain (AJ519441), and nucleotides 451 to 1,029 of the RE4 isolate from Reunion Island (AM409201). On the basis of the initial sequence obtained, specific primers (RM-TYLCV 583C: 5'-CCA CGA GTA ACA TCA CTA ACA-3' and RM-TYLCV 895F: 5'-GGA ACA GGC ATT AGT TAA GAG-3') were designed to amplify the remainder of the genomic sequence by PCR followed by cloning and sequencing of the amplicons. At least three clones were sequenced to arrive at the consensus sequence. Sequence comparisons showed that the TYLCV isolate from Mauritius had the greatest sequence identity (95 to 100%) with the above isolates. To our knowledge, this is the first report of TYLCV in tomato in Mauritius. In view of the economic importance of leaf curl disease in tomato in many parts of the world, an island-wide survey needs to be carried out to monitor the disease and assess its impact on tomato production. References: (1) M. Peterschmitt et al. Plant Dis. 83:303, 1999. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

First Report of Iris yellow spot virus in Onion in Mauritius.

Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) transmitted by thrips (Thrips tabaci Lindeman) is an economically important viral pathogen of bulb and seed onion (Allium cepa) crops in many onion-growing areas of the world (2,3). In Africa, IYSV has been reported in Reunion (4) and South Africa (1). In June 2008, diamond-shaped lesions that are typical of IYSV were observed on onion seed scapes in an onion plot of 0.25 ha at Reduit in the central part of Mauritius. Disease incidence was 80% with a severity of 50 to 75% of the scape surface area. Lodging was observed in 25% of the symptomatic plants. Twenty-two symptomatic plants were tested and found to be positive for IYSV when tested by double antibody sandwich (DAS)-ELISA with a commercially available kit (Agdia Inc., Elkhart, IN). The presence of the virus was confirmed by reverse transcription (RT)-PCR tests with primers 917L: 5'-TAAAACTTAACTAACACAAA-3' and 56U: 5'-TCCTAAGTATTCACCAT-3' as forward and reverse primers, respectively, for specific sequences flanking the CP gene. Another set of primers specific to the small (S) RNA of IYSV (5'-TAAAACAAACATTCAAACAA-3' and 5'-CTCTTAAACACATTT AACAAGCAC-3') produced an amplicon of approximately 1.2 kb that includes the 772-bp nucleocapsid (N) gene. The 1.2-kb amplicon was cloned and four clones were sequenced and consensus sequence was used for comparisons. Sequence analysis showed that the N gene of the IYSV isolate from Mauritius (GenBank Accession No. HM218822) shared the highest nucleotide sequence identity (99%) with several known IYSV N gene sequences (Accession Nos. FJ785835 and AM900393) available in the GenBank, confirming the presence of IYSV in the onion crops in Mauritius. A survey was subsequently carried out from July to November 2008 in major onion-growing localities at La Marie, Henrietta, Reduit, and Plaine Sophie (center); Bassin, La Ferme, and La Chaumiere (west); Grand Sable, Petit Sable, and Plaisance (south, southeast); and Belle Mare, Trou d'Eau Douce, and Palmar (east) to monitor the distribution of the disease on the island. Symptomatic samples with diamond-to-irregularly shaped lesions were observed and 155 symptomatic and 35 nonsymptomatic samples were collected and screened by DAS-ELISA for IYSV and Tomato spotted wilt virus (TSWV), another tospovirus reported to infect onion elsewhere. Sixty-six percent of the symptomatic samples screened (102 of 155) tested positive for IYSV. No IYSV was detected in the symptomless samples. There was no serological indication of TSWV infection in the samples. Samples that tested positive for IYSV were collected from Belle mare, Palmar, and Trou d'eau douce in the east and La Ferme in the west. Cultivars infected were Gandiole, Local Red, and Veronique. No IYSV was detected in the bulbs. The vector, T. tabaci, was observed in infected onion parcels surveyed and is known to occur in all onion-producing areas of the island. To our knowledge, this is the first report of IYSV in onion in Mauritius. Further surveys and monitoring of IYSV incidence, along with its impact on the yield, need to be established. References: (1) L. J. du Toit et al. Plant Dis. 91:1203, 2007. (2) D. H. Gent et al. Plant Dis. 88:446, 2004. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) I. Robène-Soustrade et al. Plant Pathol. 55:288, 2006.

First Report of Tomato chlorosis virus in Tomato on Mauritius Island.

In February of 2007, a virus disease survey on tomato plants (Solanum lycopersicum) in greenhouses and open fields was conducted on the island of Mauritius at the request of the Agricultural Research and Extension Unit (AREU), sponsored by the European Union, and funded by the Programme Régional de Protection des Végétaux (PRPV). Yellowing symptoms on the lower and middle leaves of tomato plants and whiteflies (Bemisia tabaci) were observed in greenhouses in Pailles, located in the north region of the island. The interveinal chlorosis pattern of the discolored leaves was similar to symptoms described for Tomato chlorosis virus (ToCV; genus Crinivirus) detected on tomato in 2004 on Reunion Island (1), suggesting the possible involvement of the same virus. Six symptomatic tomato leaf samples were collected from separate plants in the Pailles greenhouses. Total RNA was extracted from these samples with the Qiagen (Courtaboeuf, France) RNeasy Plant Mini Kit. Reverse transcription-PCR was used for molecular diagnosis, independently using two sets of specific ToCV primers. The first set of primers, ToCV-172 and ToCV-610, was designed to amplify the highly conserved region of the heat shock protein 70 (HSP70) gene (2). The second set of primers was designed to amplify the coat protein (CP) gene (forward-CP-ToCV-4384: 5'-ATCCTCTGGTTAGACCGTTAG-3' and reverse as in Segev et al. [3]). PCR products of the expected size (439 and 725 bp, respectively) were observed for the six samples from the greenhouse from Pailles. For each set of primers, two PCR products obtained from two different samples were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI) and sequenced (Macrogen, Seoul, Korea). The two HSP70 sequences (GenBank-EMBL-DDBJ Accession Nos. AM884013 and AM884014) and the two CP sequences (FM206381 and FM206382) had 100% nucleotide identities (DNAMAN; Lynnon BioSoft, Quebec City, Canada). The highest nucleotide identities of the 439-bp fragment of HSP70 gene (NCBI, BLASTn) were 97% with ToCV isolates from France (DQ355214, DQ355215, and DQ355216), Florida (AY903448), Italy (AM231038 and AY048854), Mayotte Island (AM748818), Portugal (AF234029), and Reunion Island (AM748816). Similarly, the highest nucleotide identities (98%) were obtained with ToCV isolates from France (EU625350) and Spain (DQ136146), with the 725-bp fragments of CP gene. Interestingly, ToCV isolates from Mauritius and Reunion are as divergent as isolates from the rest of the world, which suggests the possibility of different introductions. In conclusion, observed symptoms and laboratory results based on two different regions of the genome confirm the presence of ToCV in symptomatic tomatoes on the island of Mauritius, for the first time to our knowledge. The visual survey carried out in June of 2008 confirmed the presence of typical interveinal chlorosis symptoms in other greenhouses, requiring further studies to assess the incidence of ToCV on tomato crops. References: (1) H. Delatte et al. Plant Pathol. 55:289, 2006. (2) D. Louro et al. Eur. J. Plant Pathol. 106:589, 2000. (3) L. Segev et al. Plant Dis. 88:1160, 2004.

First Report in Mauritius of Bacterial Leaf Blight of Syngonium Caused by Xanthomonas campestris pv. syngonii.

In November of 2006, necrotic leaf lesions with water-soaked margins were observed on Syngonium podophyllum in Floréal, Forest Side, and Réduit, Mauritius. Although not an economically important crop, the disease was of concern because syngonium is a host for Xanthomonas axonopodis pv. dieffenbachiae and the anthurium industry is of major economic importance in Mauritius. X. campestris pv. syngonii, described as the causal agent of bacterial leaf blight of syngonium (2), is genetically closely related to group 9.4 X. axonopodis pv. dieffenbachiae strains (3). In contrast to X. axonopodis pv. dieffenbachiae, X. campestris pv. syngonii strains are highly virulent on syngonium but are not pathogenic on anthurium or other Araceae, but both react similarly to the Xcd108 monoclonal antibody (Mab) (Agdia Inc., Elkhart, IN) and to a nested PCR assay designed for X. axonopodis pv. dieffenbachiae (4). X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains can be distinguished on the basis of restriction analysis of the amplicon of this PCR assay. Four pure cultures isolated from S. podophyllum were gram negative, yellow pigmented, and produced mucoid colonies on yeast peptone glucose agar (YPGA). One positive control strain of X. campestris pv. syngonii (LMG 9055 from the United States) and X. axonopodis pv. dieffenbachiae (LMG 695 from Brazil) were also used for all tests. All strains reacted positively with the Xcd108 MAb using indirect ELISA. DNA from all strains was amplified by the nested PCR assay, and the HincII restriction pattern of the amplicons identified strains from Mauritius as X. campestris pv. syngonii. Pathogenicity tests were performed on 8-month-old plants of Anthurium andreanum cv. Florida, Dieffenbachia maculata cv. Tropic Marianne, and S. podophyllum cv. Robusta by infiltrating suspensions containing ~1 × 105 CFU ml¯1 of each strain prepared from YPGA plates. Each strain was inoculated onto three young leaves (four inoculation sites per leaf) on two plants. Negative control plants received sterile Tris buffer solution (10 mM, pH 7.2). Plants were maintained in a growth chamber with day and night temperatures at 30 ± 1°C and 26 ± 1°C, respectively, 95 ± 5% relative humidity, 30 µmol·m¯2·s¯1 light intensity, and a photoperiod of 12 h (4). All strains caused typical water-soaked lesions 14 days after inoculation (dai) on syngonium. Lesions turned necrotic with chlorotic margins 27 to 34 dai. Typical bacterial blight lesions were observed on anthurium leaves inoculated with X. axonopodis pv. dieffenbachiae strain LMG 695, but no symptoms were observed 60 dai when strains from Mauritius and LMG 9055 were used. Amplified fragment length polymorphism analysis of four strains from Mauritius and additional reference, X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains, using SacI/MspI and four primer pairs (unlabeled MspI+1 [A, C, T, or G] primers and 5'-labeled-SacI+C primer for the selective amplification step) (1), showed that the strains from Mauritius could be distinguished from X. axonopodis pv. dieffenbachiae but were identical to X. campestris pv. syngonii strains from the United States and Réunion Island. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) R. S. Dickey and C. H. Zumoff. Phytopathology 77:1257, 1987. (3) J. L. W. Rademaker et al. Phytopathology 95:1098, 2005. (4) I. Robene-Soustrade et al. Appl. Environ. Microbiol. 72:1072, 2006.