The reference strain of ‘Ca. Phytoplasma trifolii’ is the causative agent of clover proliferation (CP) disease of alsike clover (Trifolium hybridum). The CP disease was first reported in Canada in the...
The reference strain of ‘Ca. Phytoplasma trifolii’ is the causative agent of clover proliferation (CP) disease of alsike clover (Trifolium hybridum). The CP disease was first reported in Canada in the early 1960s when the aetiological agent was mistakenly presumed to be a yellows-type virus (Chiykowski, 1965). Subsequent investigations revealed that the disease was associated with infection by a mycoplasma-like organism (Chen and Hiruki, 1975; Hiruki and Chen, 1984), now termed phytoplasma, strain CPR (Hiruki and Wang, 2004). Later, phytoplasmas of the same lineage (subgroup 16SrVI-A) were found in the USA, Mexico, and many countries in Europe and Asia, causing diseases in diverse leguminous and vegetable crops, responsible for significant yield losses and quality reductions. Phytoplasmas of the same lineage also caused disease in elm trees in the USA. Phytoplasmas of closely-related lineages (various subgroups of group 16SrVI) also have wide distributions around the world.
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 CPR 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’, CPR, 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 CPR 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).
The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.
‘Ca. Phytoplasma trifolii’ and closely related phytoplasma strains in the clover proliferation group are not listed as quarantine pests by the European and Mediterranean Plant Protection Organization (EPPO).
‘Ca. Phytoplasma trifolii’ and closely related phytoplasma strains in the clover proliferation group have a broad range of hosts, affecting both dicotyledonous and monocotyledonous plants. Among commonly affected dicotyledonous hosts are agriculturally important leguminous, solanaceous and brassica crops such as lucerne, beans, clover, tomato, potato, pepper, brinjal, cabbage and mustard (Lee et al., 2004a; Lee et al., 2004b; Girsova et al., 2017; Kumari et al., 2019). Economically important monocotyledonous hosts include date palm and maize (Zibadoost et al., 2016; Zamharir and Eslahi, 2019). ‘Ca. Phytoplasma trifolii’ can also infect environmentally important ornamentals and forest trees; for the latter, an example is American elm, to which strains of ‘Ca. Phytoplasma trifolii’ can cause devastating elm yellows disease (Jacobs et al., 2003; Flower et al., 2018). It is worth noting that elm yellows disease can also be caused by another phytoplasma species, ‘Ca. Phytoplasma ulmi’.
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).
Phytoplasmas are nutritionally fastidious. There is no indication that any phytoplasma can survive outside of its host; no pure phytoplasma culture has been established in any cell-free medium thus far. In nature, like other phytoplasmas, ‘Ca. Phytoplasma trifolii’ reference strain CPR and other related strains in the clover proliferation group (16SrVI) are transmitted by phloem-feeding insect vectors. It has been demonstrated that the reference strain CPR (type member of subgroup 16SrVI-A) is transmitted from clover (Trifolium hybridum) to other plants by Macrosteles fascifrons (Chiykowski, 1965; Hiruki and Wang, 2004). In the Pacific northwest of the USA, BLTVA phytoplasma (also belonging to subgroup 16SrVI-A) has a broad range of hosts including potato, tomato, radish, carrot, sugarbeet, dry bean, groundsel, kochia and shepherd’s purse. BLTVA phytoplasma is mainly transmitted by beef leafhopper Circulifer tenellus (Crosslin et al., 2005; Munyaneza et al., 2006; Munyaneza et al., 2007; Munyaneza et al., 2010; Crosslin et al., 2012; Murphy et al., 2014). Under experimental conditions, C. tenellus was able to transmit BLTVA phytoplasma among 43 plant species belonging to at least 14 different families (Golino et al., 1989). In Mexico, subgroup 16SrVI-A phytoplasmas are reportedly transmitted by leafhoppers Ceratagallia nitidula and Empoasca abrupta (Salas-Muñoz et al., 2018). In Russia, leafhoppers Euscelis incisa and Aphrodes bicinctus have been implicated in transmitting 16SrVI-A phytoplasmas among various leguminous crops (Girsova et al., 2017). In Iran, leafhoppers Circulifer haematoceps [Neoaliturus haematoceps] and Orosius albicinctus [Orosius orientalis] are potential vectors for transmitting subgroup 16SrVI-A phytoplasmas that affect various plants including sesame, cabbage and golden marguerite (Salehi et al., 2007; Salehi et al., 2017; Hemmati et al., 2018). In India, where phytoplasmas of the subgroup 16SrVI-D are prevalent, leafhopper Hishimonus phycitis has been identified as a potential vector (Kumar et al., 2017; Gopala et al., 2018). In addition, according to earlier studies (as reviewed by Weintraub and Beanland, 2006, and references therein), Orosius argentatus, Batracomorphus punctatus and Euscelis spp. were also involved in transmitting clover proliferation group (16SrVI) phytoplasmas.
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).
Presently, the most efficient and accurate way to detect and identify ‘Ca. Phytoplasma trifolii’ is PCR-amplification of phytoplasmal 16S rRNA gene fragment and subsequent DNA sequencing of the amplicon. The sensitivity of phytoplasma detection can be enhanced by using two-step enriched-nested PCR technique. The most commonly used phytoplasma-universal primer pairs for two-step nested PCR technique PCRs include P1A/16S-SR (Lee et al., 2004c) and R16F2n/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996). After DNA sequencing of the amplicons, an iPhyClassifier analysis online (Zhao et al., 2009; https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) can help to determine whether an infecting phytoplasma is affiliated with ‘Ca. Phytoplasma trifolii’.
Field visual survey is useful for a rapid assessment of overall health conditions of the plants in the field and the extent of possible phytoplasma infections (see Symptoms). However, as mutually distinct phytoplasma species may induce similar symptoms, it is necessary to conduct laboratory diagnostic tests on field samples to confirm phytoplasma infection and to identify ‘Ca. Phytoplasma trifolii’.
Mutually distinct phytoplasma species may induce the plant disease or diseases with indistinguishable symptoms. Several plant diseases that are caused by ‘Ca. Phytoplasma trifolii’ infection can also be caused by infection of some other Candidatus phytoplasma species. For example, the aetiological agent of elm yellows disease can either be ‘Ca. Phytoplasma trifolii’ (Jacobs et al., 2003; Flower et al., 2018) or ‘Ca. Phytoplasma ulmi’ (Griffiths et al., 1999; Lee et al., 2004c). Likewise, the causative agent of potato purple top disease can either be ‘Ca. Phytoplasma trifolii’ (BLTVA, Crosslin et al., 2005) or a number of other Candidatus phytoplasma species including ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’ (Kumari et al., 2019). In such cases, laboratory tests (PCR-amplification of phytoplasmal 16S rRNA gene and subsequent DNA sequencing of the amplicon) are necessary to identify the infecting phytoplasma. The signature sequences defined in the formal descriptions of ‘Ca. Phytoplasma trifolii’ (Hiruki and Wang, 2004) will be helpful in distinguishing ‘Ca. Phytoplasma trifolii’ from other phytoplasma species.
Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.
There is no cure for phytoplasmal diseases. Good agricultural practices, such as the use of healthy plant materials, removal of diseased plants, eradication of potential phytoplasma reservoirs (weeds) and control of insect vectors, can help in preventing and managing diseases caused by ‘Ca. Phytoplasma trifolii’.
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