An epidemic of almond witches'-broom in Lebanon: Classification and phylogenetic relationships of the associated phytoplasma

An epidemic of almond witches'-broom has devastated almond production in Lebanon. Thousands of almond trees have died over the past 10 years due to the rapid spread of the disease. The symptoms, which include early flowering, stunted growth, leaf rosetting, dieback, off-season growth, proliferation of slender shoots, and witches'-brooms arising mainly from the main trunk and roots, resemble those caused by phytoplasmal infections. For the detection of the putative causal agent, nested polymerase chain reaction (PCR) was performed using universal primers (P1/P7, R16mF2/R16mR1, and R16F2n/R16R2) commonly used for the specific diagnosis of plant pathogenic phytoplasmas. Phytoplasmas were readily detected from infected trees with witches'-broom symptoms collected from three major almond growing regions in Lebanon. Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified by the primer pair R16F2n/R16R2 revealed that the phytoplasma associated with infected almonds is similar to, but distinct from, members of the pigeon pea witches'-broom phytoplasma group (16SrIX). A new subgroup, 16SrIX-B, was designated. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that almond witches'-broom (AlmWB) phytoplasma is most closely related to members of the pigeon pea witches'-broom phytoplasma group (with sequence homology ranging from 98.4 to 99.0%). Phylogenetic analysis of 16S rDNA sequences from AlmWB phytoplasma and from representative phytoplasmas from GenBank showed that the AlmWB phytoplasma represents a distinct lineage within the pigeon pea witches'-broom sub-clade. The same phytoplasma appears also to infect peach and nectarine seedlings.