P. Jones¹*, Y. Arocha², O. Antezana³, E. Montellano³ and P. Franco⁴

¹ Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK
² National Center for Animal and Plant Health (CENSA), Apdo 10, San Jose de Las Lajas, Havana, Cuba
³ Ladiplantas Community Plant Clinic, Comarapa, Bolivia
⁴ CIAT, Santa Cruz, Bolivia

*phil.jones@bbsrc.ac.uk

Accepted for publication 06/10/04

Tomatoes (Lycopersicon esculentum), an important cash crop for smallholder farmers in the hillside production systems of the Valles cruceños, Santa Cruz Province and Rio Chico, Sucre Province, Bolivia, were surveyed during 2002-3. Symptoms of hoja de perejil start with adventitious sprouting of axillary buds and rapid elongation of side shoots, which break through the crown of normal leaves (Fig. 1). Leaves of the side shoots are small and fern-like (Fig. 2) and as the season progresses large bushy plants are produced (Fig. 3). Flowers are reduced in size and do not appear to set fruit but some fruit may be produced on the early normal growth. Infected plants were screened for virus using lateral flow kits (Pocket Diagnostics®), ELISA and electron microscopy. Only Tomato mosaic virus was identified but this was not consistently associated with hoja de perejil. DNA was extracted from affected plants and tested by nested PCR for phytoplasma using generic rDNA primers P1 (Deng & Hiruki, 1991) / P7 (Schneider et al., 1995) and R16F2n/R16R2 (Gundersen & Lee, 1996). Phytoplasma products were confirmed using the endonucleases HaeIII, RsaI and AluI, and by direct sequencing of the 16S/23S spacer region (SR) with P4 (Smart et al., 1996) / P7 primers.

Figure 1: Early symptoms of hoja de perejil	Figure 2: Healthy tomato leaf (left), hoja de perejil (right)

During the tomato crop surveys, plants of Morrenia variegata (Asclepiadaceae), a vine growing in hedgerows, were found around affected tomato fields near San Rafael, Santa Cruz Province, that showed symptoms of little-leaf (Fig. 4). DNA was extracted from these plants and indexed for phytoplasma as above. Amplimers (1250 bp) with identical RFLP profiles were consistently obtained from hoja de perejil and M. variegata with little-leaf. SR sequences from hoja de perejil (GenBank Accession No. AY725208) and Morrenia little leaf (No. AY725207) were compared with those of other phytoplasmas in GenBank using BLAST. They showed a maximum (91%) homology with phytoplasmas from the 16SrI Aster yellows group.

Figure 3: Hoja de perejil: large bushy tomato plant	Figure 4: M. variegata showing little-leaf symptoms (upper, left), alongside a healthy plant (lower, right)

This is the first report of hoja de perejil disease of tomato, where infection rates of over 60% were seen in some fields of the most popular cultivar ‘Rio Grande’. This is also the first report of Morrenia little leaf disease and its association with a phytoplasma. The BLAST results suggest that the phytoplasma found associated with these diseases may belong to a new 16Sr phytoplasma group.

Work in the UK was done under Defra plant health licence no. PHL 174B/4612 (09/2003).

Deng S, Hiruki D, 1991. Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. Journal of Microbiological Methods 14, 53-61.

Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-51.

Schneider B, Seemüller E, Smart C, Kirkpatrick BC, 1995. Phylogenetic classification of plant pathogenic mycoplasmalike organisms or phytoplasmas. In: Razin R, Tully JG, eds. Molecular and Diagnostic Procedures in Mycoplasmology, Vol I, San Diego, USA: Academic Press, 369-80.

Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz KH, Seemüller E, Kirkpatrick BC, 1996. Phytoplasma-specific primers based on sequences of the 16S-23S rRNA spacer region. Applied and Environmental Microbiology 62, 2988-93.

The British Society for Plant Pathology