Molecular Evidence for the Presence of Maize Bushy Stunt Phytoplasma in Corn in Brazil

Previously, electron microscopy revealed that the corn (Zea mays L.) disease characterized by stunting and leaf reddening and commonly known as "red stunt" in Brazil is associated with plant infection by an unidentified phytoplasma (formerly mycoplasmalike organism) (1). During recent years, corn production in Brazil has been seriously affected by the increasing prevalence of a disease exhibiting symptoms similar to those of "red stunt." The present investigation was initiated to determine the possible association of a phytoplasma with the current disease problem and to attain definitive molecular identification of any associated phytoplasma. To detect the possible presence of a phytoplasma in diseased corn in Brazil, plants exhibiting symptoms of stunting and leaf reddening in the field in 1995 and 1996 were assayed for the presence of phytoplasma DNA sequences by the use of polymerase chain reactions (PCR). We used primer pairs R16mF2/R16mR1 and R16F2n/R16R2 in nested PCRs (2) to prime phytoplasma-universal amplification of 16S ribosomal (r) DNA. Oligonucleotide pair rpF1/rpR1 (3) was used to prime phytoplasma-universal amplification of ribosomal protein (rp) gene operon sequences. Phytoplasma identification was accomplished by restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA and rp gene operon sequences. Primer pair MBS-F1/MBS-R1 (4) was used to prime amplification of a maize bushy stunt (MBS)-specific chromosomal DNA sequence. Preparation of template DNAs, PCR conditions, and RFLP analyses of PCR products were as previously described (2-4). DNA amplification was observed in all PCRs containing template DNAs derived from symptomatic plants, indicating phytoplasmal infection of corn in Brazil. No DNA amplification was observed in PCR containing template DNA from healthy control corn plants. Polymorphisms in amplified 16S rDNA were those characteristic of phytoplasmas classified in 16S rRNA gene group 16SrI, subgroup I-B, of which MBS phytoplasma is a member (3). Collective RFLP patterns of amplified rp gene operon sequences were similar or identical to those observed in parallel tests for a known reference strain of MBS phytoplasma, indicating that the Brazilian corn plants were infected by MBS phytoplasma. Amplification of MBS-characteristic DNA was observed in PCRs containing MBS-specific primer pair MBS-F1/MBS-R1 and DNA from diseased corn, confirming infection of the plants by MBS phytoplasma. This work provides the firs