Candidatus Phytoplasma trifolii
(clover proliferation phytoplasma)

Preferred Common Name

• clover proliferation phytoplasma

Other Scientific Names

• Phytoplasma trifolii

International Common Names

• English: alfalfa witches'-broom phytoplasma; beet leafhopper-transmitted agent (BLTVA); potato purple top phytoplasma; potato witches'-broom phytoplasma; tomato big bud phytoplasma

EPPO code

• PHYPTR

• Domain: Bacteria
•     Phylum: Firmicutes
•         Class: Mollicutes
•             Order: Acholeplasmatales
•                 Family: Acholeplasmataceae
•                     Genus: Phytoplasma
•                         Species: Candidatus Phytoplasma trifolii

Phytoplasmas are cell wall-less bacteria that infect vascular plants. Morphologically, phytoplasmas resemble animal- and human-infecting mycoplasmas therefore once had a trivial name, mycoplasma-like organism. On the basis of phylogenetic analysis of near full-length 16S rRNA gene sequences, phytoplasmas constitute a monophyletic clade within the Mollicutes (Gundersen et al., 1994) and are most closely related to members of the genus Acholeplasma within the Anaeroplasma clade (Weisburg et al., 1989). Despite decades of numerous attempts and recent progress (Contaldo et al., 2019), sustained axenic culture has not been demonstrated for any phytoplasma thus far. According to the convention for recording properties of uncultured organisms (Murray and Schleifer, 1994; Murray and Stackebrandt, 1995), a provisional genus ‘Candidatus Phytoplasma’ was erected to accommodate phytoplasmas (The International Research Programme for Comparative Mycoplasmology Phytoplasma/Spiroplasma Working Team - Phytoplasma Taxonomy Group, 2004). This provisional genus is embraced within the class Mollicutes, order Acholeplasmatales. ‘Candidatus Phytoplasma trifolii’ is one of the taxa established in the provisional genus and it was named after the plant host in which it was discovered, clover (Trifolium hybridum). Clover proliferation phytoplasma CP^R is the reference (type) strain of the taxon (Hiruki and Wang, 2004).

In addition to Candidatus species assignment, phytoplasmas are also classified into groups and subgroups based on RFLP analysis of a 1.25 kb PCR-amplified 16S rDNA segment (F2nR2 fragment) using a defined set of 17 restriction enzymes (Lee et al., 1993; Lee et al., 1998). The phytoplasma groups delineated using this classification scheme are consistent with 16S rRNA gene phylogeny (Zhao et al., 2009). The 16S rDNA RFLP markers further separate phytoplasma strains in the same group into subgroup lineages. The phytoplasma 16S rDNA RFLP analysis also evolved from the original actual enzymatic approach to DNA sequence-based computational simulation (Wei et al., 2007; Zhao et al., 2009; Zhao and Davis, 2016). The reference strain of ‘Candidatus Phytoplasma trifolii’, CP^R, is the initial member of the clover proliferation group, subgroup A (16SrVI-A) (Lee et al., 2000; Hiruki and Wang, 2004).

It was conceived that each phytoplasma 16Sr group should represent at least one species (Gundersen et al., 1994). Presently, ‘Ca. Phytoplasma trifolii’ is the only species recognized in the group 16SrVI. As each of the known members in the existing subgroups of 16SrVI shares >97.5% similarity with CP^R in their 16S rDNA sequences, all current members in the group 16SrVI are considered ‘Ca. Phytoplasma trifolii’-related strains in this datasheet.

Like other phytoplasmas, ‘Ca. Phytoplasma trifolii’ resides in phloem sieve cells of infected plants and is transmitted from infected plants to healthy ones through phloem-feeding insects, mainly leafhoppers, planthoppers and psyllids. Phytoplasmas do not have a clearly defined shape (being amorphous); they may appear ovoid, oblong or filamentous under electron microscope. The size of phytoplasma cells ranges from 0.2 to 0.8 µm in diameter (Doi et al., 1967; McCoy et al., 1989; Weintraub and Beanland, 2006).

Infection by ‘Ca. Phytoplasma trifolii’ or other phytoplasma strains in the clover proliferation group induces a wide variety of symptoms in hosts. Symptom types differ depending on host species and stages of infection. Most frequently observed symptoms during vegetative growth stage include shoot proliferation, witches’-broom growth, little leaf, leaf yellowing and stunting. Prominent floral symptoms include virescence, phyllody, big bud and flower multiplication. Results from studies on potato purple top (PPT) phytoplasma (subgroup 16SrVI-A) infection in tomato revealed a profound disruption of gibberellin homeostasis in host plants (Ding et al., 2013a; Ding et al., 2013b). It was discovered that PPT phytoplasma infection can derail plant meristem cells from their genetically pre-programmed destiny, therefore altering the growth and developmental pattern of the host plant (Wei et al., 2013). Studies revealed that PPT phytoplasma was able to induce multiple symptoms in a single tomato plant sequentially - a total of eight mutually distinct symptoms (witches'-broom growth, disrupted sympodial growth pattern, cauliflower-like inflorescence, big bud, virescence, floral organ duplication, parthenocarpy and vivipary) were identified. Each symptom was linked to a stage-specific event of apical meristem destiny derailment and corresponding transcriptional reprogramming (Wei et al., 2013; Wei et al., 2019).

In addition to inducing visible symptoms (morphological changes), ‘Ca. Phytoplasma trifolii’ infection can also induce profound physiological changes in host plants. Studies revealed that a pepper-infecting strain (also a member of subgroup 16SrVI-A) was able to alter the metabolic activities of the host, resulting in a progressive increase in an array of secondary metabolites including phenolic compounds, flavonoids, condensed tannins and anthocyanins (Reveles-Torres et al., 2018b); the phytoplasma infection also reduced CO₂ fixation, decreased invertase activity, inhibited glycolysis, and altered sugar and amino acid compositions in the host tissues (Velásquez-Valle et al., 2019).

Vector Transmission (Biotic)

In nature, ‘Ca. Phytoplasma trifolii’, including other closely related 16SrVI phytoplasma strains, is transmitted by phloem-feeding insect vectors. Macrosteles fascifrons and Circulifer tenellus are confirmed major vectors (Golino et al., 1989; Hiruki and Wang, 2004; Munyaneza et al., 2006). Other potential vectors include Aphrodes bicinctus, Batracomorphus punctatus, Ceratagallia nitidula, Circulifer haematoceps [Neoaliturus haematoceps], Empoasca abrupta, Euscelis incisa, Hishimonus phycitis, Orosius albicinctus [Orosius orientalis] and O. argentatus (Weintraub and Beanland, 2006; Salehi et al., 2007; Girsova et al., 2017; Kumar et al., 2017; Salehi et al., 2017; Gopala et al., 2018; Hemmati et al., 2018; Salas-Muñoz et al., 2018).

Natural Dispersal

Generally, abiotic factors are not involved in natural dispersal of phytoplasmas. However, it has been noted that atmospheric structure and motions could influence insect migration (Drake and Farrow, 1988). Low-level jet stream wind-facilitated long-range movement of phytoplasma-infected aster leafhopper (M. fascifrons) was reported (MacRae, 2014) although ‘Ca. Phytoplasma trifolii’ was not specifically mentioned.

Accidental Introduction

‘Ca. Phytoplasma trifolii’ is not seed transmittable but can be spread through propagules such as tubers, runners, bulbs and cuttings. The pathogen may be accidentally introduced into new areas by moving vegetatively propagated plant materials that are already infected but are asymptomatic.

‘Ca. Phytoplasma trifolii’ can also be transmitted through graft union from an infected component to a healthy component.

‘Ca. Phytoplasma trifolii’ and other phytoplasma strains in the clover proliferation group (16SrVI) cause diseases in a variety of agriculturally important plant species including leguminous, solanaceous and brassica crops, inflicting commodity yield losses and quality reductions. Economic impacts vary year to year and location to location depending largely on population dynamics of the transmission vectors. Taking BLTVA phytoplasma-induced potato purple top disease in the Pacific northwest of the USA as an example, one study revealed that there was a mean decrease in potato tuber yield of “0-12% at a density of one beef leafhopper per plant, 6-19% at two beet leafhoppers per plant, and 6-20% for five beet leafhoppers per plant” (Murphy et al., 2014).

Draft datasheet under review