First report of a ‘Candidatus Phytoplasma asteris’-related strain associated with little leaf disease of Helianthus debilis in Florida, USA

N. A. Harrison^* and E. E. Helmick

University of Florida, Plant Pathology Dept., Research and Education Center, 3205 College Avenue, Fort Lauderdale, FL 33314, USA

^*naha@ufl.edu

Accepted for publication 26/02/08

Beach sunflower (Helianthus debilis) with symptoms indicative of phytoplasma disease were observed in a demonstration garden of native plants in Broward county, Florida during 2005, and again, in 2006.  Symptoms observed on plants included stunting, shoot proliferation, apical bunches of small, yellowed leaves (Fig. 1A) and floral abnormalities (Fig. 1B).  

Figure 1:  Beach sunflower (Helianthus debilis) plants displaying pronounced
little leaf (A) and floral abnormality (B) symptoms.

DNA samples extracted from shoots of 11 diseased and two symptomless plants were assessed for phytoplasma DNA by a PCR assay (35 cycles) employing phytoplasma universal primer pair P1 (Deng & Hiruki, 1991) and P7 (Smart et al., 1996).  After products of initial PCRs were diluted (1:25) with sterile water and reamplified (30 cycles) by PCR incorporating nested 16S rRNA gene primer pair R16mF2/R16R1 (Gundersen & Lee, 1996), an rDNA amplicon of about 1.4 kb in size was consistently obtained from all 11 diseased plants and from one of two symptomless plants.  Comparison of restriction fragment profiles, after digestion of nested rDNA products with DdeI, MseI or RsaI endonuclease, revealed no differences among phytoplasmas associated with little leaf diseased (LL) or symptomless (HD) H.  debilis.  DdeI profiles (Fig.  2A) failed to distinguish LLHD phytoplasma from aster yellows (16SrI) group phytoplasma reference strains maize bushy stunt (MBS) and Florida periwinkle virescence (FPV).  However, RsaI (data not shown) or MseI (Fig.  2B) profiles each differentiated LLHD phytoplasma from 16SrI group reference strains eastern aster yellows (EAY) and strawberry green petal (SGP). 

Figure 2:  Restriction fragment length polymorphism (RFLP) analysis of phytoplasma rDNA products derived from little leaf diseased beach sunflower (Helianthus debilis) plants by nested PCR employing rRNA primer pairs P1/P7 followed by R16mF2/R16R1.  RFLP patterns of rDNA after digestion with DdeI (A) or MseI (B) endonuclease.  Samples for comparison consisted of diseased beach sunflower plants (HD1-HD7) and phytoplasmas associated with eastern aster yellows (EAY), maize bushy stunt (MBS), Florida periwinkle virescence (FPV) or strawberry green petal (SGP) in Florida.

Phylogenetic analysis of 16S rDNA sequences identified LLHD phytoplasma (GenBank Accession No.  DQ381828) as a ‘Candidatus Phytoplasma asteris’-related strain most closely related to MBS and to phytoplasma strains associated with diseases of dogfennel (Eupatorium capillifolium) (DQ381534), ragweed (Ambrosia artemisiifolia) (DQ321822) and periwinkle (Catharanthus roseus) (DQ381535) that occur in southern Florida.  This is a first report of a new disease of H. debilis attributed to a ‘Ca.  Phytoplasma asteris’-related strain in Florida.   

References

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

Gundersen DE, Lee I-M,  1996.  Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs.  Phytopathologia Mediterranea 35, 144-151.

Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz K-H, Seemüller E, Kirkpatrick BC, 1996.  Phytoplasma-specific PCR primers based upon sequences of the 16-23S rRNA spacer region.  Applied and Environmental Microbiology 62, 2988-2993.