Biology and chemistry of an Umbravirus like 2989 bp single stranded RNA as a possible causal agent for Opuntia stunting disease (engrosamiento de cladodios) - A Review

Abstract

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. It was 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 the
strongly symptomatic cactus with this disease. As almost all plant viruses go through a
replication phase involving double stranded RNA (dsRNA), a dsRNA extraction was
performed and a dsRNA species of about 600 bp identified. Then, reverse-transcribed the
dsRNA, amplified the resultant cDNA by PCR, and cloned and sequenced the 600 bp
fragment that were 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. Primers made from this 630
bp fragment were used to extend this sequence to 2989 sequence. This sequence appears to
be a full-length sequence with three open reading frames (ORF) and is shorter than the
closest class of viruses, the Umbraviruses that can be spread by mechanical transmission
and by aphids. It was not possible to transmit the virus or symptoms mechanically. Over a
six-year period using traditional PCR, this virus was found in hundreds of symptomatic cacti
but not in non-symptomatic pads. RT-PCR has found low levels of this virus on nonsymptomatic
cladodes (3.7 fg) on a symptomatic plant and much higher concentrations
(1x102 to 1x105 fg) on symptomatic cladodes from the same plant. Black bean aphids (Aphis fabae), that are the vector for a closely related Umbravirus known as groundnut rosetta virus,
have been routinely found on the unopened flowers of cactus. This Umbravirus was found in
aphids feeding on symptomatic cladodes. As Umbraviruses cannot infect plants without a
companion Luteovirus, that provides the protein coat for the Umbravirus, degenerate
Luteovirus primers were used and a probable incomplete Luteovirus-like 4797 bp sequence
was found on aphids feeding on symptomatic cactus. This Luteovirus was not found in
Opuntia cladodes using PCR. A micro RNA assembly of six pooled symptomatic Opuntias
did not find a contig that spanned the 4797 putative Luteovirus sequence, but some
fragments as large as 44 bp were exact matches to the Luteovirus. As Umbraviruses occur
throughout the plant but Luteoviruses only occur in the phloem, lower Luteovirus
concentrations would be expected. Two successive one hour 60°C heat treatments
eliminated these symptoms on new growth that was also PCR negative. A 5839 bp
Potexvirus was found in some of these cladodes but its presence was not correlated with any
symptoms. Similar symptomatic cacti in Italy, South Africa and Mexico should be examined
with these primers and dsRNA to see if similar correlations between presence/absence of this
fragment and symptomatic plants can be obtained. It is suggested that this disease be known
as OSD (Opuntia Stunting Disease).