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In November 2001, a leaf curl disease of tomato, manifested by yellowing of leaf lamina, upward leaf curling, leaf distortion, shrinking of leaf surface, and stunted plant growth was observed in tomato-growing areas in the Varanasi and Mirzapur districts of eastern Uttar Pradesh, India, which caused yield losses up to 100%. The causal agent was infective to tomato cv. Punjab Chuhara by whiteflies and grafting. Inoculated plants developed symptoms observed in naturally infected tomatoes. Viral DNA was isolated from artificially inoculated tomato plants using 1% CTAB (2) followed by a concentration of supercoiled DNA by alkaline denaturation (1). A geminivirus was confirmed by polymerase chain reaction using DNA-A degenerate primers (3), and a 550-bp amplified product was obtained from artificially and naturally infected plants. Full-length viral genomes of DNA-A and DNA-B were cloned in plasmid pUC18 at HindIII and XbaI sites, respectively. Partial tandem dimers of the viral clones were infective to Nicotiana benthamiana and tomato cv. Organ Spring through particle bombardment. Infected N. benthamiana plants exhibited downward and upward leaf curling, big veins, leaf puckering with interveinal chlorosis, and stunting. On tomato, symptoms were the same as those seen on naturally infected plants. Cloned DNA also infected Capsicum annuum cv. California Wonder (upward leaf curling and stunting) and tobacco cv. Xanthi (leaf curling and crinkling) but failed to infect Phaseolus vulgaris, okra, cotton, and N. glutinosa. The Varanasi isolate was sap transmissible (0.1 M potassium phosphate buffer, pH 7.0) from the bombarded plants to N. benthamiana and tomato cv. Organ Spring. DNA-A alone infected N. benthamiana (upward leaf curling and big veins) and tomato cv. Organ Spring (mild leaf curl), but symptoms were delayed and milder. Full-length genome sequencing revealed DNA-A (AY190290) contained 2,757 nt and DNA-B (AY190291) contained 2,688 nt. DNA-A of the Varanasi isolate shares 98.4% identity with a DNA-A sequence (AF449999) obtained from a tomato showing leaf curl symptoms from the same region and 97.1% identity with an isolate from Gujarat (900 km from Varanasi). All three sequences represent isolates of the same species, herein called Tomato leaf curl Gujarat virus, based on the priority of submission of the DNA sequence for the Gujarat region (ToLCGV; AF 413671). All isolates noted were obtained from GenBank. However, except for the DNA-A sequence, no other information is available for these ToLCGV isolates. DNA-A of the ToLCGV-Varanasi isolate shares 66.8% identity with Tomato leaf curl New Delhi virus, severe strain (ToLCNdV-Svr) (U15015), and 84.1% with Tomato leaf curl Karnataka virus (U38239). No DNA-B has been reported for these two ToLCGV isolates, and no infectious clone proving the etiology of the disease has been constructed, except for ToLCGV-Varanasi. DNA-B of ToLCGV-Varanasi shares 79.2% homology with ToLCNdV-Svr and 84.1% with ToLCNdV-Luc (X89653). These results suggest that the isolate from Varanasi belongs to ToLCGV, a previously undescribed geminivirus species causing a devastating tomato leaf curl disease in Gujarat and Uttar Pradesh. References: (1) H. C. Birnboim and J. Doly. Nucleic Acids Res. 7:1513, 1979. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Whitefly-transmitted geminivirus diseases cause important losses in several horticultural crops in all areas in Mexico (1). Tomatillo is important in the Mexican diet since it is widely used to prepare many types of salsas and other dishes. As a result, tomatillo, also known as tomate verde (green tomato), is cultivated in 29 of 32 states in Mexico, with the main production areas located in the states of Morelos, Puebla, and Michoacán. Leaf samples of 105 tomatillo plants exhibiting yellowing, yellowing mosaic, leaf curl, bunchy top, and stunting were collected from the states of Puebla, Morelos, Estado de México, and Sinaloa. Symptomatic plants were associated with the presence of whiteflies in many fields and suggested a viral etiology. Total DNA extracted from symptomatic tomatillo plants was used as a template in a polymerase chain reaction (PCR)-based geminivirus detection procedure. MP16 and MP82 primers (2) were used to direct the amplification of a segment from the stem-loop structure in the intergenic region (IR) to a conserved region in the coat protein (CP) of begomoviruses (2). Sixty-nine percent (72/105) of the samples produced the expected PCR fragment (400 to 450 bp). Similar results were obtained with a dot-blot hybridization procedure using as a probe the component A of Pepper huasteco virus (PHV) under low stringency conditions. More than 50 PCR products were cloned and sequenced. Sequence analysis (nucleotide level for the IR; amino acid level for the CP) revealed that the tomatillo-infecting geminiviruses clustered into two main groups. The first group showed a high percent identity (average of 95.3% at the CP N terminus) to PHV, whereas the second showed a similarly high percent (average 93.8%) identity to Pepper golden mosaic virus (PepGMV, previously called Texas pepper geminivirus. Both PepGMV and PHV were found in all sampled areas. Although mixed infections (differentiated by the respective IR probes) of PHV and PepGMV were common (61%), single infections were also detected (PHV 27%; PepGMV 10%). The presence of begomoviruses in tomatillo crops has been previously reported (1); however, their identity as PHV and PepGMV was not confirmed. References: (1) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996. (2) P. Umaharan et al. Phytophatology 88:1262, 1998.

Cassava mosaic disease (CMD) occurs in all cassava-growing regions of Africa, India, and Sri Lanka. Characterized by mosaic and distortion of cassava leaves and reduced plant growth, causing high yield losses, CMD is caused by geminiviruses (genus Begomovirus, family Geminiviridae) transmitted through infected cuttings or by the whitefly, Bemisia tabaci. Three such geminiviruses have been described: African cassava mosaic virus (ACMV) occurs in most of the cassava-producing zones of Africa; East African cassava mosaic virus (EACMV) in East Africa; and Indian cassava mosaic virus (ICMV) in the Indian subcontinent (1). The two components of ACMV and ICMV genomes, DNA-A and DNA-B, have been sequenced; only DNA-A of EACMV has been identified and sequenced. Variations in symptom expression and severity within the same cassava variety have been observed in Cameroon. To determine the nature of the virus species inducing such variations, 50 samples were collected from CMD-infected plants in the savannah and rainforest zones of Cameroon: 2 from the sahel/savannah plain, 13 from the western highland savannah, and 35 from the main cassava-producing belt of the southwestern rainforest. There is a high incidence of CMD in the rainforest region, with some farms completely infected, while in the savannah regions farms generally have less than 25% incidence. Variation in symptom expression was more common in the rainforest region. Samples were collected from plants with distinct symptoms and/or different extents of symptom severity, then analyzed with the polymerase chain reaction (PCR) with specific primers: JSP1, ATG TCG AAG CGA CCA GGA GAT; JSP2, TGT TTA TTA ATT GCC AAT ACT; and JSP3, CCT TTA TTA ATT TGT CAC TGC. Primer JSP1 anneals to the 5' end of the coat protein (CP) of ACMV and EACMV; primers JSP2 and JSP3 anneal to the 3' ends of ACMV and EACMV, respectively. Virus identification was based on presence of an amplified fragment of either virus. ACMV was detected in all 50 samples; EACMV was detected in 8. All samples infected with EACMV were from the southwestern rainforest of Cameroon and were more severely affected by the disease than single infected plants. Previous reports have limited occurrence of EACMV to East Africa (1). This is the first report of the occurrence of EACMV in West Africa. The CP gene of three isolates of EACMV from Cameroon (EACMV/CM) was sequenced from cloned PCR products. There was a high CP nucleotide sequence identity (>99%) with only two amino acid differences among all three EACMV isolates. In contrast, there was a rather low sequence identity (94%) with EACMV/TZ from Tanzania (2), suggesting they may belong to a previously undescribed West African strain of EACMV. This indicates the geminiviruses causing CMD in Africa are more widely distributed than previously reported. None of the Cameroon isolates showed the type of recombination of the EACMV isolate from Uganda (EACMV/ UG) (having the CP core segment the identical to the corresponding ACMV CP sequence) (2). This emphasizes the need for characterization of the viruses causing CMD in different cassava-growing regions of Africa since appropriate control strategies depend on adequate knowledge of disease etiology. References: (1) Y. G. Hong et al. J. Gen. Virol. 74:2437, 1993. (2) X. Zhou et al. J. Gen. Virol. 78:2101, 1997.