First Report of Aster Yellows Phytoplasma in Alfalfa

Abstract

Samples of alfalfa (Medicago sativa L.) leaves and stems showing symptoms of inter-veinal chlorosis and purpling, commonly associated with insect feeding, were collected from 8 sites in central and southern Wisconsin in autumn of 1998. Samples were frozen within 24 h of collection. Approximately 0.3 g of plant tissue from each sample was used for total DNA extraction according to the protocol of Zhang et al. (4), with minor modifications in grinding procedures and reagent volumes to optimize results. Nested polymerase chain reaction (PCR) was carried out by amplification of 16S rDNA with the universal primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 as described by Gunder-sen and Lee (1). Undiluted total sample DNA was used for the first amplification; PCR products were diluted (1:30) in sterile water prior to final amplification. Alfalfa DNA and sterile water were used as negative controls; DNA from phytoplasma causing X-disease in peach (CX) served as a positive control. Fragments of 16S rDNA from putative phytoplasmas amplified by PCR with the primer pair R16F2n/R16R2 were characterized by restriction endonuclease digestion (3). The resulting restriction fragment length polymorphism (RFLP) patterns were compared with patterns for known phytoplasmas described by Lee et al. (3). Products of nested PCR were also purified and sequenced with primers R16F2n/R16R2 and an automated DNA sequencer (ABI 377XL; C. Nicolet, Biotechnology Center, University of Wisconsin-Madison). Of 51 samples of alfalfa assessed, one sample from Evansville, WI, yielded a nested PCR product of the appropriate size (1.2 kb), indicating the presence of phytoplasma. Digestion of this product with various restriction enzymes produced RFLP patterns that were identical to those for phytoplasmas in the aster yellows phytoplasma subgroup 16SrI-A (3). Alignment of the DNA sequence of the nested PCR product from the positive sample with sequences found in the GenBank sequence data base (National Center for Biotechnology Information, Bethesda, MD) with the BLAST sequence similarity function confirmed this result. Although other phytoplasma strains (particularly those causing witches'-broom) have been reported to infect alfalfa (2), this is the first report of the presence of the aster yellows phytoplasma in the alfalfa crop. Vectors involved in transmission and the potential agronomic impacts of aster yellows phytoplasma in alfalfa are topics of current investigation. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A.-H. Khadhair et al. Microbiol. Res. 152:269, 1997. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) Y.-P. Zhang et al. J. Virol. Methods 71:45, 1998.

Identification of aster yellows phytoplasmas in soybean in Wisconsin based on RFLP analysis of PCR-amplified products (16S rDNAs)

“…Our hypothesis was that phytoplasmas have expanded their host range in Wisconsin to include legume crops. Detection of phytoplasmas in alfalfa in Wisconsin has been reported previously (Peters et al 1999). …”

“…Alfalfa is a known host of phytoplasmas in Wisconsin (Peters et al 1999). Plants were maintained in single rows spaced 76 cm apart and 30 plants/m of row.…”

Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

“…Phytoplasma group 16SrII was also previously reported to infect faba bean in Sudan (Alfaro-Fernández et al 2012) and alfalfa with little leaf and witches' broom in Iran and Oman, respectively (Khan et al 2002;Hosseini et al 2013). Phytoplasma group 16SrI was associated with alfalfa plants in Bolivia, Lithuania and the USA (Peters et al 1999;Jomantiene et al 2000;Jones et al 2005). Although there are many reports about onion infection by phytoplasma group 16SrI around the world (Khadhair et al 2002;Lee et al 2003;Jomantiene et al 2010), the association of 16SrII phytoplasma with onion is considered as the first report not only in Saudi Arabia, but also in the world.…”

Characterization of phytoplasmas related to peanut witches’-broom and stolbur groups associated with alfalfa diseases in Iran

“…Several phytoplasmas belonging to different groups were recognized as causal agents of alfalfa witches'-broom disease. In America, phytoplasmas related to 'Candidatus Phytoplasma asteris' are known to cause witches'-broom disease in alfalfa in the USA (Peters et al 1999) and Bolivia (Jones et al 2005). Similarly, 'Ca.…”