Genetic Diversity and Geographical Distribution of Phytoplasmas Associated with Potato Purple Top Disease in Mexico

Potato purple top (PPT) disease has caused severe economic losses in some potato (Solanum tuberosum) growing areas of Mexico. Two distinct phytoplasma strains belonging to the aster yellows and peanut witches'-broom groups (16SrI and 16SrII groups) have been associated with PPT disease in several regions of Mexico. However, there has been no previous large-scale survey in the main potato growing areas in Mexico to analyze the diversity and geographical distribution of phytoplasmas. Potato samples were collected between 2003 and 2006 and were analyzed by nested polymerase chain reaction assays. On the basis of results from nucleotide sequence comparisons and virtual restriction fragment length polymorphism analysis of 16S rDNA, four different phytoplasma groups were detected in potato growing areas in Mexico. The aster yellows group (16SrI) ‘Candidatus Phytoplasma asteris’ was distributed in all potato growing areas, whereas peanut witches'-broom group (16SrII) ‘Candidatus Phytoplasma aurantifolia’ was detected in Guanajuato and Sinaloa, X-disease group (16SrIII) was detected in Coahuila and Guanajuato, and the Mexican periwinkle virescence (16SrXIII) was only detected in Sinaloa. Phytoplasmas from X-disease and Mexican periwinkle virescence groups were detected in potato samples for the first time in Mexico.

First Report of a Defect of Processing Potatoes in Texas and Nebraska Associated with a New Phytoplasma

An outbreak of a new potato disease occurred in Texas and Nebraska causing a serious defect in potato chips produced from commercial processing potatoes. The defect consists of patchy brown discoloration of chips and can be a cause for rejection of contracted potatoes by the processor. Infected potato plants exhibit symptoms of the purple top wilt syndrome similar to those of the purple top disease in processing potatoes caused by clover proliferation phytoplasma recently found in Washington and Oregon (3). Foliar symptoms include stunting, chlorosis, slight purple coloration of new growth, swollen nodes, proliferated axillary buds, and aerial tubers. Tuber symptoms include mild vascular discoloration and brown flecking of medullary rays. Seed potatoes from affected plants produce hair sprouts. Total nucleic acid was extracted from leaf and stolon tissue of symptomatic plants in the field and from tuber samples exhibiting the defect from commercial storages. Nested polymerase chain reactions (PCR) were performed using phytoplasma-universal 16SrDNA-based primers (P1/P7 and R16F2n/R16R2) (2) to detect the presence of phytoplasmas in these samples. A negative control, devoid of DNA templates in the reaction mix, was included in all PCR assays. In 2004, 13 foliar samples tested positive for phytoplasmas using PCR. None of the apparently symptomless plants or tubers tested positive. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA using enzymes AluI, MseI, HhaI, BfaI, and Tsp509I indicated that four samples are associated with a phytoplasma belonging to subgroup A (16SrI-A) of the “Candidatus Phytoplasma asteris” (aster yellows phytoplasma) group (16SrI), and nine plant samples were associated with a new phytoplasma related to, but distinct from, the stolbur phytoplasma group (16SrXII). Nucleotide sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. DQ174114-DQ174123) confirmed the results on the basis of RFLP analyses. Sequences of cloned 16S rDNAs were analyzed with previously described phytoplasma strains available in GenBank using DNAStar's (Madison, WI) Lasergene software MegAlign program. The new phytoplasma is only distantly related to the stolbur phytoplasma, sharing 96.6% sequence homology. In 2005, 14 defective tuber samples from storage and 16 symptomatic plants from the field tested positive for the new phytoplasma. In Texas and Nebraska, it appears that at least two distinct phytoplasmas seem to be involved in the disease complex contributing to the defects of processed products produced from infected potatoes. Previous reports have suggested a similar defect of chipping potatoes, but the phytoplasma associated with the disease was not identified (1). To our knowledgek, this the first report of this new phytoplasma associated with disease and defects of potato and the first report of this phytoplasma in the United States. References: (1) E. E. Bantarri et al. Trans. ASAE 33:221, 1990. (2) I.-M. Lee et al. Int. J. Sys. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Plant Dis. 88:429, 2004.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Potato Purple Top Disease in Washington and Oregon

An epidemic of purple top disease of potato (Solanum tuberosum) occurred in the Columbia Basin Region of Washington and Oregon in 2002 and 2003, causing great economic loss in the potato industry (1). Symptoms of potato purple top (PPT) were characterized by upright terminal shoots, upward leaf rolling, chlorosis, red or purplish discoloration of new leaves, proliferation of axillary shoots with basal swelling, and the formation of aerial tubers. Preliminary studies on PPT disease suggested phytoplasma as a possible cause (1). In this study, 78 potato samples (including five asymptomatic) were collected from five fields throughout the region. A nested polymerase chain reaction (PCR) with primer pair P1/P7 in the first amplification followed with primer pair R16F2n/R16R2 was performed to detect the presence of phytoplasmas in infected plants (2). Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Eighty-four percent (63% in the first amplification) of the symptomatic samples and 60% (0% in the first amplification) of the asymptomatic samples tested positive. Low phytoplasma titers and the presence of PCR inhibitors accounts for the low detection rate in the first PCR amplifications. RFLP analyses of 16S rDNA with enzymes MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma associated with PPT belonged to the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) (2). 16SrVI-A currently consists of three members, CP (GenBank Accession No. AY500130), potato witches'-broom (GenBank Accession No. AY500818), and vinca virescence (VR) (GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (2). The taxonomic affiliation of PPT phytoplasma was confirmed by phylogenetic analysis of cloned 16S rDNA (GenBank Accession Nos. PPT4, AY496004; PPT8, AY496005). The 16S rDNA sequences of the PPT strains were closely related to VR with 99.7% sequence homology compared with 99.2% with CP. A high correlation between the symptoms and the presence of 16SrVI-A phytoplasmas in the potato plants suggests that these phytoplasmas play an etiological role in PPT disease. To gain further evidence, a modified test of Koch's postulates was conducted. Infected tissues from four phytoplasma-positive potato samples (including PPT4 and PPT8) were grafted onto healthy potato seedlings. Within 60 days after grafting, the potato seedlings developed symptoms similar to those in the original diseased samples. The newly infected plants were maintained through cuttings. RFLP analysis of 16S rDNA indicated that the phytoplasmas detected in each of the seedlings and cuttings were identical to those in the scions. These results confirmed the probable etiological role of CP group, subgroup 16SrVI-A phytoplasma strains in PPT disease in Washington and Oregon. There are two other confirmed cases of phytoplasmas (BLTVA and aster yellows phytoplasma) associated with PPT disease in Utah (4) and Mexico (3). References: (1) P. B. Hamm et al. Potato Prog. Vol. 3, No. 1, 2003. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) N. E. Leyva-Lopez et al. Can. J. Microbiol. 48:1062, 2002. (4) C. D. Smart et al. Phytopathology 83:1399, 1993.