PROGRESO EN LA IDENTIFICACIÓN DEL AGENTE CAUSAL DE "ENGROSAMIENTO DEL CLADODIO O MACHO"

Perhaps the most economically important disease of Opuntia ficus indica fruit cacti in Mexico is the engrosamiento de cladodios or macho disease. The symptoms of this disease, which has been suggested to be caused by a phytoplasma, are severe stunting of cladodes, flowers and fruits. In the mid 1980s this disease appeared in commercial cactus fruit orchards of D’Arrigo Bros near Gonzalez, California. We performed more than 30 PCR-based tests for viruses as well as various extraction methods and polymerase chain reaction (PCR) tests for phytoplasmas but were unable to find any of the known viruses or mycoplasmas in our strongly symptomatic cactus with this disease. As almost all plant viruses go through a replication phase involving double stranded RNA (dsRNA), we performed a dsRNA extraction and identified a dsRNA species of about 600 bp. We then reverse-transcribed the dsRNA, amplified the resultant cDNA by PCR, and cloned and sequenced the 600bp fragment corresponding to the 600 bp dsRNA species that we had identified in symptomatic tissue. When this sequence was compared to translated DNA in the National Center for Biotechnology Information (NCBI) nucleotide data base (BLAST analysis) it was most similar to the Tobacco bushy top virus (E score of 2e- 39 ), which is a single stranded RNA virus with no DNA intermediate. This virus falls in the category of an umbravirus that can be spread by mechanical transmission and by aphids. We have found cowpea aphids (Aphis craccivora), that are the vector for a closely related umbravirus known as groundnut rosetta virus, on the unopened flowers of cactus. By use of RT-PCR analysis with primers that were designed to amplify the 600 bp fragment, we have this RNA in 16 out of 17 symptomatic plants and in none of 13 non-symptomatic plants. We suggest that similar symptomatic cacti in Italy, South Africa and Mexico be examined by extracting dsRNA and performing RT- PCR with these primers to see if similar correlations between presence/absence of this fragment and symptomatic plants can be obtained.