DETECTION AND IDENTIFICATION OF PHYTOPLASMA DNA IN SYMPTOMATIC MUSHROOMS OF THE GENUS RAMARIA (O. GOMPHALES)

TELLERIA (2003). Detection and identification of phytoplasma DNA in symptomatic mushrooms of the genus Ramaria (O. Gomphales). Bot. 60(1): 11-18. Using polymerase chain reaction assays with specific primers for amplifying phytoplasma 16S rDNA, the presence of phytoplasmas in Ramaria basidiomes with abnormal development has been detected for the first time in fungi. Phytoplasmas have not been detected in asymptomatic basidiomes. Sequence analyses based on parsimony and maximum likelihood place the phyto plasma in the Stolbur group.


INTRODUCTION
Phytoplasmas are wall-less bacteria in the class Mollicutes (formerly called mycoplasmalike organisms) associated with diseases of many perennial or herbaceous crops worldwide (LHERMINIER & al., 1999). They have a wide host range and are mostly restricted to phloem tissue. As phytoplasmas are recalcitrant to cultivation, little is known of their biology. They are specially vectored by phloem-feeding insects in which they multiply (BOUDON-PADIEU & al., 1989). During the last decade, DNA-based methods of identification have shown that phytoplasmas form a unique clade within mollicutes (SEEMULLER & al., 1994). According to LHERMINIER & al. (1999), to control the phytoplasma diseases more information is necessary on hostpathogen relationships and on the replication strategies of phytoplasmas. It has been suggested that herbaceous plants could act as a phytoplasma reservoir when the main host has disappeared.
Most species included in the genus Ramaria (O. Gomphales, Basidiomycotina: JULICH, 1981;HAWKSWORTH & al., 1995) are ectomycorrhizal and they have strong fleshy and coralloid basidiomes, branching several times from the base. In general, the branch surface is smooth and basidiome apices are small and delicate, clearly distinct from the branches. Occasionally basidiomes of the ectomycorrhizal species show an abnormal growth: a) the branches become wrinkled; b) the ramification rank decreases with the branches enlarging and fusing together, and c) the apices are indistinct with the terminal branch blunted and inflated ( fig. 1). However, under light microscope, no abnormal features were seen in the hyphae or spores.
Comparison of these symptoms in Ramaria with those observed in many plants at the Laboratori de Sanitat Agraria in Barcelona (Spain) suggested some kind of disease caused by a virus or phytoplasma infection. Thus, a study was undertaken to verify the presence of these pathogens in the Ramaria species with the symptoms described above. Because of their specificity and sensitivity, during the last two years we have been focusing on molecular methods to detect and identify phytoplasmas in plants based on PCR (AHRENS & SEEMULIJER, 1992). According to LEE & al. (1998) and SEEMOLLER & al. (1998) in phytoplasmas the 16S rDNA is a very useful region to separate taxa at the species or genus level.

MATERIAL AND METHODS
DNA from less that 10 mg of healthy and diseased Ramaria basidiomes (table 1)  PCR amplifications. Nested-PCR assays were performed with the primer pair P1/P7 (DENG & HIRUKI, 1991;SMART & al., 1996) followed by the primer pair fU5/rU3 (LORENZ & al., 1995)   was used as template in nested-PCR. The PCR reactions were performed by using a Techne DNA thermocycler (model Progene). The cycling parameters were 40 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min with a final extension at 72 °C for 10 min. Before initiating the cycling parameters an initial denaturation at 94 °C for 5 min was done. Controls, lacking DNA, were run for each experiment to check for DNA contamination of the reagents.
DNA sequencing. Amplification products were cleaned using the E.Z.N.A. Clean kit (Omega Biotech) and both strands were sequenced separately using primers fU5 and rU3 with an ABI Prism 377 genetic Analyzer and the ABI Prism™ BigDye™ terminator Cycle Sequencing Ready Reaction kit with AmpliTaq®DNA Polymerase (Perkin Elmer Applied Biosystem, Norwalk, Connecticut, USA). Sequence Navigator™ Sequence Comparison software (Perkin Elmer) was used to identify the consensus sequence from the two strands of each ITS region.
To control for possible contamination of extractions, amplifications and sequencing reactions were replicated at the Laboratori de Sanitat Agraria (Barcelona) and at the Real Jardi'n Botanico (Madrid) following the same protocols as mentioned above, but independent reagents and a thermocycler Perkin Elmer 9700.
Analyses of data. The nucleotide sequence obtained (FTTOPL_l) has been logged in the EMBL database and has been compared with 59 sequences retrieved from the EMBL Gen-Bank (table 2). To search for the best alignment Sequence Navigator™ Sequence Comparison for pairwise comparisons and SEQAPP software for multiple sequences were used. Where ambiguities in the alignment occurred, the alignment chosen was the one generating the fewest potentially informative characters. Alignment gaps were marked "-" and unresolved nucleotides or unknown sequences were indicated with "N".
Parsimony analysis was performed using the computer program PAUP 4.0b* (Phylogenetic Program Using Parsimony) of SWOF-FORD (1998). Clostridium innocuum and Acholeplasma laidlawii were used as outgroup. Branch robustness was estimated by bootstrap analysis (FELSENTEIN, 1985) of 10,000 heuristic replicates using the fast stepwise-addition option. In the maximum likelihood analysis (ML) (FELSENSTEIN. 1981) optimality criterion was employed.

RESULTS
No PCR products were visualized with primers P1/P7 from any of the collections analyzed. With fU5/rU3 no amplifications were obtained from asymptomatic collections. The four symptomatic samples give amplifications of 850 bp, in agreement with the expected size, but only from FITOPL_l was the concentration enough for sequencing. Moreover, in FITOPL_2 and FITOPL_4 an extra band about 800 bp was visualized.
The sequence length obtained is 800 bp and has been logged in the NCBI with the Accession Number AJ430066. The sequences of the 60 taxa were aligned to produce a matrix of 826 characters, of which 203 were parsimony-informative.
Two hundred most parsimonious trees were obtained under heuristic search with a tree length of 723 steps, CI = 0.6321, RI = 0.8845, RC = 0.5591. In the strict consensus tree ( fig. 2) the sequence obtained cluster with phytoplasmas from Stolbur group with 99 % bootstrap support. The topology of the ML tree agrees with the consensus tree inferred from heuristic search ( fig. 3).
The highly sensitive nested-PCR combined with the use of specific primers has made possible the detection of phytoplasmas in symptomatic basidiomes of Ramaria. The absence of visible products from samples amplified by direct PCR with P1/P7 is probably due to the low concentration of these prokaryotes as described in plant hosts (BERTACCINI &al., 1996).
Plants infected by phytoplasmas exhibit an array of symptoms that suggest profound disturbance in the normal balance of plant hormones or growth regulators. The stolbur phy-toplasma is an ubiquitous agent that multiplies in a broad range of cultivated and wild plant species (GARNIER, 2000). The symptoms change according to the host, some affected plants shown variegate branches, as observed in olive-trees (TORRES & MARTIN, unpublished). These symptoms are the same as detected in Ramaria spp. and fit with the molecular analysis, that includes phytoplasmas found in this fungus as belonging to the stolbur group. These phytoplasmas are very frequent in wild plants found in infected grapevines. BATLLE & al. (2000) identified stolbur phytoplasmas in Convolvulus arvensis, Lavandula officinalis, Polygonum con-volvulus, Solarium nigrum and Thymus officinalis. According to our molecular analysis, stolbur group phytoplasmas are the sister group of the Aster yellows phytoplasma clade.