Detection, Identification and Molecular Characterization of a 16SrII-V subgroup Phytoplasma Associated with Nicotiana plumbaginifolia Viviani

Nicotiana plumbaginifolia Viviani or commonly known as curl-leaved tobacco is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani found in Yunlin County, Taiwan was observed to have phyllody, virescence, and witches’-broom which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches’-broom phytoplasma, a ‘Candidatus phytoplasma aurantifolia’-related strain. Further identification of SAP54/PHYL1 and SAP11 homologues in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches’-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease which poses threat to the agricultural sector and food security in Taiwan.

QPCR detection and quantification of ‘Candidatus phytoplasma solani’ in tomato with primers targeting CPN60 gene

Tomato is one of the most economically important crops in Republic of Moldova. However, it is affected by a number of pathogens. One of the wide spread diseases is stolbur caused by the infection agent ‘Candidatus Phytoplasma solani’. Accurate diagnostics of the disease at an early stage is essential for successful control of the disease. In this work, we describe detection and quantification of ‘Candida-tus Phytoplasma solani’ in tomato by real-time PCR, as well as suitability of the method for assessing resistance of different tomato varieties to Phytoplasma infection.

Multilocus Genotyping of ‘Candidatus Phytoplasma solani’ Associated with Rubbery Taproot Disease of Sugar Beet in the Pannonian Plain

Rubbery taproot disease of sugar beet (RTD), associated with ‘Candidatus Phytoplasma solani’, appeared in 2020 on an epidemic scale in northern Serbia and southern Slovakia, situated at opposite edges of the Pannonian Plain. In the affected locations where the disease was assessed, symptomatic sugar beets were analysed for phytoplasma infection. Additionally, multilocus sequence analyses of ‘Ca. P. solani’ strains on epidemiologically informative marker genes (tuf, stamp and vmp1) were performed. Symptomatic sugar beets from other countries of the Pannonian Plain (Croatia, Hungary and Austria), one sample from Germany, and red beets from Serbia were included in the analyses. ‘Ca. P. solani’ was detected in sugar beet in all assessed countries, as well as in red beet. Molecular analyses revealed the high genetic variability of ‘Ca. P. solani’ with the presence of all four tuf-types (a, b1, b2 and d), 14 stamp genotypes (seven new) and five vmp1 profiles (one new). The most common multilocus genotype in Serbia, Slovakia, Croatia, and Hungary was dSTOLg (tuf-d/STOL/V2-TA). It was dominant on sites with epidemic RTD outbreaks in the Pannonian Plain and in several sugar beet fields with non-epidemic RTD occurrence suggesting the prevalence of a particular epidemiological pathway during the epidemic’s phases.

Novel effector recognition capacity engineered into a paired NLR complex

The Arabidopsis RRS1-R Resistance gene confers recognition of the bacterial acetyltransferase PopP2 and another bacterial effector, AvrRps4. The RRS1-S allele recognizes AvrRps4 but not PopP2. RRS1-R/RRS1-S heterozygotes cannot recognize PopP2. RRS1-R and RRS1-S also suppress the constitutive RPS4-dependent autoactivity of RRS1-Rslh1. Phytoplasmas cause important plant diseases, and their effectors can cause degradation of specific host proteins. We tested whether attaching a pathogen effector-dependent degron to RRS1-R, enabling its degradation by phytoplasma effector SAP05, could derepress RRS1-Rslh1 autoactivity, resulting in SAP05-dependent resistance. In transient assays in tobacco, RRS1-R-derived constructs can confer a hypersensitive response (HR) to SAP05. However, phytoplasma infection assays in transgenic Arabidopsis resulted in delayed disease symptoms but not full resistance. We provide a proof-of-concept strategy utilizing the recessiveness of a plant immune receptor gene to engineer recognition of a pathogen effector that promotes degradation of a specific host protein.

Biochemical changes induced by phytoplasmas in plants.

Phytoplasmas are cell wall-less and pleomorphic bacterial pathogens that cause considerable yield losses in more than 1000 plant species worldwide and transmitted by sap-feeding insects. Phytoplasma-infected plants exhibit symptoms including witches' broom, phyllody, virescence, yellowing induced by the phytoplasma effector proteins. It has been revealed that phytoplasmas are capable of modulating biochemical responses of plants involved in defense mechanisms. In this respect, phenolic compounds, free amino acids in source leaves, protein contents, some secondary metabolites, and polyamines were found to increase, while chlorophyll content and photosynthetic activity and the number of chemical compounds were reduced as a consequence of phytoplasma infection. Therefore, the objective of this study was to review the chemical composition changes in phytoplasma-infected plants.

Microscopic studies of the phytoplasma infection effect on the symbiotic system Medicago sativa – Rhizobium meliloti in simulated conditions

Aim. Aim of the study was to microscopically investigate the interaction of Acholeplasma laidlawii var. granulum 118 with the target plant Medicago sativa, as well as to determine the role of Rhizobium meliloti strains with natural and defective polysaccharides in the plant’s resistance occurrence. Methods. Cultivation of plants and their inoculation with bacteria was performed under conditions of microvegetation experiment. The study of the ultrastructure of alfalfa roots and nodules was carried out with both light and electron microscopy. Results. The rhizobial mutant strain, defective in the synthesis of lipopolysaccharides, more often formed atypical nodules on M. sativa, which aged faster. In the variant with double inoculation of alfalfa with rhizobia together with acholeplasma changes in the morphology of the lateral roots of plants, as well as deformation of the nodules were observed. Conclusions. Results of this study indicate not only the ability of phytoplasmas to penetrate the root system and migrate to plant aboveground organs, but also demonstrate the possibility of their presence in the nodules formed by rhizobia. Keywords: Mollicutes, acholeplasma, rhizobia, mutant, lipopolysaccharides.

Application of Plant Defense Elicitors Fails to Enhance Herbivore Resistance or Mitigate Phytoplasma Infection in Cranberries

Synthetic elicitors of the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways can be used to increase crop protection against herbivores and pathogens. In this study, we tested the hypothesis that elicitors of plant defenses interact with pathogen infection to influence crop resistance against vector and nonvector herbivores. To do so, we employed a trophic system comprising of cranberries (Vaccinium macrocarpon), the phytoplasma that causes false blossom disease, and two herbivores—the blunt-nosed leafhopper (Limotettix vaccinii), the vector of false blossom disease, and the nonvector gypsy moth (Lymantria dispar). We tested four commercial elicitors, including three that activate mainly SA-related plant defenses (Actigard, LifeGard, and Regalia) and one activator of JA-related defenses (Blush). A greenhouse experiment in which phytoplasma-infected and uninfected plants received repeated exposure to elicitors revealed that both phytoplasma infection and elicitor treatment individually improved L. vaccinii and L. dispar mass compared to uninfected, untreated controls; however, SA-based elicitor treatments reduced L. vaccinii mass on infected plants. Regalia also improved L. vaccinii survival. Phytoplasma infection reduced plant size and mass, increased levels of nitrogen (N) and SA, and lowered carbon/nitrogen (C/N) ratios compared to uninfected plants, irrespective of elicitor treatment. Although none of our elicitor treatments influenced transcript levels of a phytoplasma-specific marker gene, all of them increased N and reduced C/N levels; the three SA activators also reduced JA levels. Taken together, our findings reveal positive effects of both phytoplasma infection and elicitor treatment on the performance of L. vaccinii and L. dispar in cranberries, likely via enhancement of plant nutrition and changes in phytohormone profiles, specifically increases in SA levels and corresponding decreases in levels of JA. Thus, we found no evidence that the tested elicitors of plant defenses increase resistance to insect herbivores or reduce disease incidence in cranberries.

Transcriptome and DNA Methylome Reveal Insights Into Phytoplasma Infection Responses in Mulberry (Morus multicaulis Perr.)

To reveal whether the response of mulberry to phytoplasma infection is associated with genome-wide DNA methylation changes, the methylome and transcriptome patterns of mulberry in response to phytoplasma infection were explored. Though the average methylation level of the infected leaves showed no significant difference from that of healthy leaves, there were 1,253 differentially methylated genes (DMGs) and 1,168 differentially expressed genes (DEGs) in the infected leaves, and 51 genes were found simultaneously to be differently methylated and expressed. It was found that the expression of G-type lectin S-receptor-like serine/threonine protein kinase gene (Mu-GsSRK) was increased, but its methylation level was decreased in the pathogen-infected or salicylic acid (SA)-treated leaves. Overexpression of Mu-GsSRK in Arabidopsis and in the hairy roots of mulberry enhanced transgenic plant resistance to the phytoplasma. Moreover, overexpression of Mu-GsSRK enhanced the expressions of pathogenesis-related protein 1, plant defensin, and cytochrome P450 protein CYP82C2 genes in transgenic plants inoculated with pathogens, which may contribute to the enhanced disease resistance against various pathogens. Finally, the DNA methylation dynamic patterns and functions of the differentially expressed and methylated genes were discussed. The results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection.

Knocking out AtCALS7 disturbs photoassimilate distribution and weakens defence against phytoplasma infection

Callose accumulation around sieve pores, under control of Callose synthase 7 (AtCALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. AtCALS7 is also involved in sieve-pore development and, hence, in mass-flow regulation, carbohydrate metabolism and distribution, and plant growth. Multiple roles of AtCALS7 during phytoplasma infection were investigated in healthy and phytoplasma-infected [Chrysanthemum Yellows (CY)-phytoplasma] wild-type and Atcals7ko Arabidopsis lines. In keeping with an increased phytoplasma titre in Atcals7ko plants, floral stalk height of infected wild-type and mutant plants was reduced by, respectively, 88 and 100% in comparison to their healthy controls, suggesting a higher investment of host resources in phytoplasma growth in the absence of AtCALS7. The apparently increased susceptibility of mutants was investigated by microscopic, metabolic and molecular analyses. Infection influenced the sieve-pore functionality in wild-type plants, which hardly affected plant growth, and plasmodesmata in the cortex, a phenomenon less prominent in mutants. Infection also increased the level of some sugars (glucose, sucrose, myoinositol), but to the highest extent in mutants. Finally, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) in mutants and wild-type plants and an upregulation of carbohydrate transmembrane transporters (AtSWEET11, AtSTP13, AtSUC3) in mutants only. A more effective plasmodesmal closure seemingly suppressed spread of phytoplasma effectors, which rendered wild-type plants less susceptible to infection, because gene expression of enzymes channeling carbohydrates towards phytoplasmas is less promoted

Spatiotemporal and Quantitative Monitoring of the Fate of “Candidatus Phytoplasma Solani” in Tomato Plants Infected by Grafting

Understanding how phytoplasmas move and multiply within the host plant is fundamental for plant–pathogen interaction studies. In recent years, the tomato has been used as a model plant to study this type of interaction. In the present work, we investigated the distribution and multiplication dynamics of one strain of “Candidatus Phytoplasma (Ca. P.) solani”, (16SrXII-A) in tomato (Solanum lycopersicum L., cv. Micro-Tom) plants. We obtained infected plants by grafting, a fast and effective method to maintain phytoplasma infection. In planta spread and multiplication of “Ca. P. solani” was monitored over time using qualitative and quantitative qPCR. Root, apical shoot, lower leaves, and upper leaves were sampled at each sampling time. We hypothesized that “Ca. P. solani” from the grafting site reached firstly the highest leaf, the apex and the roots; subsequently, the phytoplasmas spread to the rest of the upper leaves and then progressively to the lower leaves. Significant differences were found in “Ca. P. solani” titer among different plant tissues. In particular, the concentration of phytoplasma in the roots was significantly higher than that in the other plant compartments in almost all the sampling dates. Since the roots show rapid colonization and the highest concentration of phytoplasmas, they represent the ideal tissue to sample for an early, sensitive and robust diagnosis.