Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus

Abstract

The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity, and insect vector. Capulavirus, an eighth genus, has been proposed to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus, is shown to be transmitted by Aphis craccivora. This is the first report of an aphid-transmitted geminivirus.

FIG 1

Virions of euphorbia caput-medusae latent virus purified from agroinfected Nicotiana benthamiana plants as described previously (10) with the following modifications. The liquid N₂-powdered tissues were resuspended in the grinding buffer and homogenized in a blender for 1 min. The aqueous phase was recovered from the chloroform treatment by centrifugation, filtered through three layers of cheesecloth, and pelleted through a 10% sucrose cushion. The resuspended pellets were clarified with one low-speed centrifugation and loaded on a 10% to 40% sucrose gradient which was centrifuged at 35,000 rpm for 3 h 45 min in a Beckman SW41 rotor. The gradients were fractionated in 1-ml samples, and the fractions containing the opalescent zone of the gradient were pelleted for 2.5 h at 55,000 rpm in a Beckman Ti70 rotor. The micrograph was produced with a Jeol JEM-1400Plus transmission electron microscope.

FIG 2

(A) Genome organization of capulaviruses: alfalfa leaf curl virus (ALCV) from France, euphorbia caput-medusae latent virus (EcmLV) from South Africa, and French bean severe leaf curl virus (FbSLCV) from India. GenBank accession numbers are given in brackets. (B) Genome-wide percentage pairwise identities of capulaviruses calculated using SDT version 1.2 (11). (C) Neighbor-joining phylogenetic tree of representative geminiviruses based on full genomes. Given that it is not possible to generate a credible alignment of the genomes of all geminiviruses and that interspecies and intragenus recombination has played a significant role in geminivirus diversification, this phylogenetic tree is not intended to be accurate but is rather provided as a guide to display the degrees of sequence similarity shared by the different major groups of geminiviruses. Branches with less than 60% bootstrap support have been collapsed, and the tree is midpoint rooted.

FIG 3

Maximum likelihood phylogenetic trees of the Rep and CP amino acid sequences of representative geminiviruses together with the three known capulavirus species. Branches with less than 80% aLRT branch support have been collapsed. The Rep maximum likelihood phylogenetic tree is rooted with gemycircularviruses (12), whose Rep sequences are distantly related to those of geminiviruses, whereas that of the CP is midpoint rooted.

FIG 4

Faba bean and alfalfa plants infected with Alfalfa leaf curl virus (ALCV). Agroinfected faba bean plants exhibiting vein thickening (A) were used as source plants for transmitting ALCV to faba bean and alfalfa plants using adult Aphis craccivora. (B) Adult and larval A. craccivora. (C and D) The aphid-infected faba plants exhibited vein thickening (C) and the infected alfalfa plants, leaf curl (D). (E) A typical geminivirus DNA profile was revealed by Southern blotting of total DNA extracted from an aphid-infected faba bean plant, using P³²-labeled ALCV probes. ss, single-stranded; sc, supercoiled; oc, open circular; def, defective heterogeneous-length DNA. (F and G) Leaves of noninoculated faba bean (F) and alfalfa (G) plants are shown as controls.