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Cucumber plants (Cucumis sativus) displaying symptoms of phyllody, sterility, and flower virescence were observed in fields located in the Dianbai district of Guangdong province, China. Total DNA was extracted from the symptomatic plants; this was followed by molecular detection using a set of universal phytoplasma primer pairs, R16mF2/mR1, P1/P7, and SecYF1(II)/SecYR1(II). This resulted in the PCR amplification of products corresponding to expected sizes of 1.4 kb, 1.8 kb and 1.7 kb, respectively. The 16S rDNA sequence obtained exhibited 100% similarity with the eggplant phyllody phytoplasma, the 'Cleome rutidosperma' witches' broom phytoplasma, and the 'Desmodium ovalifolium' witches' broom phytoplasma strain DeOWB, all of which belong to the 16SrII group. Phylogenetic analysis, based on the 16S rDNA gene and SecY gene sequences, confirmed the close affiliation of the detected phytoplasma isolate, tentatively designated as cucumber phyllody phytoplasma (CuPh) China isolate, with the 16SrII-A subgroup. Additionally, virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA sequence revealed a pattern that was identical to that of the 16SrII-A subgroup. This is the first report of cucumber phyllody phytoplasma in China. [ABSTRACT FROM AUTHOR]

The bacterial genus Wolbachia induces reproductive abnormalities in its insect host, including cytoplasmic incompatibility (CI), which causes embryonic death in the crossing of infected males and uninfected females. Hence, Wolbachia-based strategies are employed to control insect pests. However, Wolbachia does not naturally infect Matsumuratettix hiroglyphicus (Matsumura), the main vector of the phytoplasma causing the sugarcane white leaf (SCWL) disease. In this study, the wYfla Wolbachia strain, which induces strong CI in its original host, was microinjected into nymphs of M. hiroglyphicus. Molecular detection revealed that Wolbachia was successfully transinfected into the recipient host, with an infection frequency of 55–80% in up to eight generations after transinfection. Wolbachia exhibited no significant detrimental effects on the developmental time of the immature stages, adult emergences, and female longevity, whereas the lifespan of transinfected males was decreased. Reciprocal crossing revealed that Wolbachia infection did not affect the number of eggs laid per female. However, the hatching rate produced by the pairs between the transinfected males and naturally uninfected females significantly decreased. The evidence of Wolbachia transmitted through the generations tested and partial CI occurrence in transinfected M. hiroglyphicus highlights the possibility of the future development of Wolbachia-based strategies for controlling the vector of SCWL. [ABSTRACT FROM AUTHOR]

Coconut (Cocos nucifera L.) is an important palm species that serves as the mainstay of several industries and contributes to the livelihoods of millions of smallholder farmers. International exchange of coconut germplasm has been undertaken for several decades to facilitate the conservation of selected varieties within global genebanks and for the distribution to farmers and scientists. In vitro systems are a convenient and an efficient method for the exchange of coconut germplasm. However, it is possible that these tissue culture systems can transfer lethal pathogens causing a threat to the importing countries. In this review, the following topics are discussed: the major disease-causing agents of concern, the various tissues that could be used for coconut germplasm exchange, and the techniques available for the detection and elimination of disease-causing agents from various transmission systems. Additionally, the lack of clear, science-backed guidelines to facilitate the exchange of in vitro coconut materials is raised, along with recommendations for future studies to ensure the safe movement of coconut germplasm without biosecurity risks. [ABSTRACT FROM AUTHOR]

Abstract Background Little leaf disease caused by phytoplasma infection is a significant threat to eggplant (also known as brinjal) cultivation in India. This study focused on the molecular characterisation of the phytoplasma strains and insect vectors responsible for its transmission and screening of brinjal germplasm for resistance to little leaf disease. Results Surveys conducted across districts in the Tamil Nadu state of India during 2021–2022 showed a higher incidence of phytoplasma during the Zaid (March to June), followed by Kharif (June to November) and Rabi (November to March) seasons with mean incidence ranging from 22 to 27%. As the name indicates, phytoplasma infection results in little leaf (reduction in leaf size), excessive growth of axillary shoots, virescence, phyllody, stunted growth, leaf chlorosis and witches’ broom symptoms. PCR amplification with phytoplasma-specific primers confirmed the presence of this pathogen in all symptomatic brinjal plants and in Hishimonus phycitis (leafhopper), providing valuable insights into the role of leafhoppers in disease transmission. BLAST search and phylogenetic analysis revealed the phytoplasma strain as “Candidatus Phytoplasma trifolii”. Insect population and disease dynamics are highly influenced by environmental factors such as temperature, relative humidity and rainfall. Further, the evaluation of 22 eggplant accessions revealed immune to highly susceptible responses where over 50% of the entries were highly susceptible. Finally, additive main effect and multiplicative interaction (AMMI) and won-where biplot analyses identified G18 as a best-performing accession for little leaf resistance due to its consistent responses across multiple environments. Conclusions This research contributes essential information on little leaf incidence, symptoms, transmission and resistance profiles of different brinjal genotypes, which together ensure effective and sustainable management of this important disease of eggplants.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP–F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

Background: Little leaf disease caused by phytoplasma infection is a significant threat to eggplant (also known as brinjal) cultivation in India. This study focused on the molecular characterisation of the phytoplasma strains and insect vectors responsible for its transmission and screening of brinjal germplasm for resistance to little leaf disease. Results: Surveys conducted across districts in the Tamil Nadu state of India during 2021–2022 showed a higher incidence of phytoplasma during the Zaid (March to June), followed by Kharif (June to November) and Rabi (November to March) seasons with mean incidence ranging from 22 to 27%. As the name indicates, phytoplasma infection results in little leaf (reduction in leaf size), excessive growth of axillary shoots, virescence, phyllody, stunted growth, leaf chlorosis and witches' broom symptoms. PCR amplification with phytoplasma-specific primers confirmed the presence of this pathogen in all symptomatic brinjal plants and in Hishimonus phycitis (leafhopper), providing valuable insights into the role of leafhoppers in disease transmission. BLAST search and phylogenetic analysis revealed the phytoplasma strain as "Candidatus Phytoplasma trifolii". Insect population and disease dynamics are highly influenced by environmental factors such as temperature, relative humidity and rainfall. Further, the evaluation of 22 eggplant accessions revealed immune to highly susceptible responses where over 50% of the entries were highly susceptible. Finally, additive main effect and multiplicative interaction (AMMI) and won-where biplot analyses identified G18 as a best-performing accession for little leaf resistance due to its consistent responses across multiple environments. Conclusions: This research contributes essential information on little leaf incidence, symptoms, transmission and resistance profiles of different brinjal genotypes, which together ensure effective and sustainable management of this important disease of eggplants. [ABSTRACT FROM AUTHOR]

The leafhopper Matsumuratettix hiroglyphicus is the vector of the phytoplasma associated with sugarcane white leaf disease in Asia. However, highly virulent strains of entomopathogenic fungi to control this vector are lacking. To select effective fungal strains, serial passaging through the leafhopper and the phenotypic properties and virulence of four strains were evaluated and compared to those of their initial stock. The influence of fungal passaging through the insect varied according to the fungal genus, strain, and passage number. The third and fifth passages of all fungal strains improved the examined parameters and showed virulence enhancement compared to those of the initial stock; however, a decrease in the parameters was observed after the seventh passage. The fifth passage of Beauveria bassiana SUTTU01 exhibited phenotypic changes resulting in significantly higher pathogenicity (80% mortality) than its corresponding initial stock. When the effect of fungal treatment on the transmission capacity of M. hiroglyphicus was tested, no significant difference was found in white leaf disease-related phytoplasma infection between untreated and fungi-treated leafhoppers. However, the fifth passage of B. bassiana SUTTU01-treated leafhoppers exhibited the lowest titer of the phytoplasma, resulting in a reduced transmission rate and fewer infected plants. This is the first report on the influence of B. bassiana on leafhopper vector capacity. The results obtained constitute a basis for future research on effective fungal strains able to reduce the phytoplasma transmission rate of this leafhopper vector. [ABSTRACT FROM AUTHOR]

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, results in the most damage to sugarcane plantations. Some SCWL canes can grow unnoticed through the maturation phase, subsequently resulting in an overall low sugar yield, or they can be used accidentally as seed canes. In this work, 12-month-old SCWL and asymptomatic canes growing in the same field were investigated. An abundance of phytoplasma in SCWL canes affected growth and sugar content as well as alterations of transcriptomic profiles corresponding to several pathways that responded to the infection. Suppression of photosynthesis, porphyrin and chlorophyll metabolism, coupled with an increase in the expression of chlorophyllase, contributed to the reduction in chlorophyll levels and photosynthesis. Blockage of sucrose transport plausibly occurred due to the expression of sugar transporters in leaves but suppression in stalks, resulting in low sugar content in canes. Increased expression of genes associated with MAPK cascades, plant hormone signaling transduction, callose plug formation, the phenylpropanoid pathway, and calcium cascades positively promoted defense mechanisms against phytoplasma colonization by an accumulation of lignin and calcium in response to plant immunity. Significant downregulation of CPK plausibly results in a reduction in antioxidant enzymes and likely facilitates pathogen invasion, while expression of sesquiterpene biosynthesis possibly attracts the insect vectors for transmission, thereby enabling the spread of phytoplasma. Moreover, downregulation of flavonoid biosynthesis potentially intensifies the symptoms of SCWL upon challenge by phytoplasma. These SCWL sugarcane transcriptomic profiles describe the first comprehensive sugarcane–phytoplasma interaction during the harvesting stage. Understanding molecular mechanisms will allow for sustainable management and the prevention of SCWL disease—a crucial benefit to the sugar industry. [ABSTRACT FROM AUTHOR]

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Parasitic plants represent a peculiar group of semi- or fully heterotrophic plants, possessing the ability to extract water, minerals, and organic compounds from other plants. All parasitic plants, either root or stem, hemi- or holoparasitic, establish a vascular connection with their host plants through a highly specialized organ called haustoria. Apart from being the organ responsible for nutrient extraction, the haustorial connection is also a highway for various macromolecules, including DNA, proteins, and, apparently, phytopathogens. At least some parasitic plants are considered significant agricultural pests, contributing to enormous yield losses worldwide. Their negative effect is mainly direct, by the exhaustion of host plant fitness and decreasing growth and seed/fruit formation. However, they may pose an additional threat to agriculture by promoting the trans-species dispersion of various pathogens. The current review aims to summarize the available information and to raise awareness of this less-explored problem. We further explore the suitability of certain phytopathogens to serve as specific and efficient methods of control of parasitic plants, as well as methods for control of the phytopathogens. [ABSTRACT FROM AUTHOR]

In surveys conducted from 2020 to 2022, five leaf samples each from symptomatic Agele marmelos trees and seedlings, along with five samples from asymptomatic trees and seedlings, were collected in Ayodhya, Uttar Pradesh, India. The DNA extraction from all the samples was subjected to nested PCR assays, using the universal phytoplasma-specific primers set (P1/P7 followed by R16F2n/R16R2). The resulting 1.2 kb amplified products were observed in all the symptomatic samples but not in the asymptomatic samples. Bael phytoplasma strain sequences from the trees and seedlings were found 100% identical within themselves and only two representative sequences (one each from tree and seedling) were deposited in GenBank (NCBI) as PP415872 (AmA-1) and PP415873 (AmA-2). BLASTn searches revealed the maximum (100%) sequence identity with a phytoplasma strain from murraya little leaf strain of Faizabad (GenBank Acc.no. OP984129) and lowest (99.84%) with arecanut crown choking of Shimoga (GenBank Acc. no. OM417502) from Karnataka. Phylogenetic analysis clustered the bael phytoplasma isolates with peanut witches' broom group phytoplasma strains. Virtual RFLP analysis confirmed their identity as 'Ca. P. australasiaticum', a 16SrII-D subgroup strain. This study presents the first identification of a phytoplasma strain in A. marmelos, emphasizing its potential threat to fruit crops and the need for vigilance in nursery practices to prevent further dissemination. [ABSTRACT FROM AUTHOR]

In summer 2018, extensive phytosanitary checks occurred in Tufanbeyli, Adana, Turkey, in a vast forest. These were prompted by significant juniper tree losses with unusual shoot growth and "witches' broom" appearance. To investigate the presence of phytoplasma in the affected juniper trees, nested-PCR assays were employed. Universal phytoplasma-specific primer pairs were used for amplification of the 16S rRNA gene. A total of 300 samples were collected, comprising 242 samples from the witches' brooms displaying symptoms and 58 samples from apparently healthy trees. PCR results showed 90.5% of symptomatic juniper trees tested positive for phytoplasma. All asymptomatic junipers, except three trees, tested negative for phytoplasma in PCR analysis. The nucleotide sequences of 107 PCR amplicons derived from the infected samples were analyzed, revealing a high degree of similarity, ranging from 99.1 to 99.3%, to the 16S ribosomal DNA (rDNA) sequence of Ligustrum witches' broom phytoplasma (LiWBP). It is worth noting that this phytoplasma has been previously documented to infect California privet (Ligustrum ovalifolium) and maize (Zea mays L.) plants in Turkey. Phylogenetic analysis of 16S rDNA identified separate LiWBP strains from Ligustrum, maize, and juniper, forming a distinct clade. This underscores their unique evolutionary lineage and genetic divergence from other known phytoplasma groups. BLAST and in-silico RFLP analysis suggest the presence of a new subgroup or lineage specific to juniper within the LiWBP-related strains. This study unveiled an emerging witches' broom disease in juniper, attributing it to the Ligustrum witches' broom phytoplasma (LiWBP) and a newly discovered subgroup lineage. [ABSTRACT FROM AUTHOR]

Simple Summary: The planthopper Pentastiridius leporinus is the main vector of two bacterial pathogens (Arsenophonus and stolbur phytoplasma) that cause a disease known as syndrome basses richesses (SBR) in sugar beet, reducing the yield and sugar content. In 2022, P. leporinus nymphs were also found to transmit Arsenophonus in potato fields, causing symptoms like wilting, yellow leaves, and rubbery tubers. We monitored both pathogens in Southwest Germany in 2022 and 2023, revealing that P. leporinus adults can transmit Arsenophonus and stolbur to potatoes. The broad prevalence of Arsenophonus was maintained, whereas the prevalence of stolbur increased in most regions in 2023. Neither of the pathogens influenced the germination rate of potato tubers, and no abnormal growth was observed after germination. Arsenophonus was not detected in germinated shoots, but stolbur was present, emphasizing the need for plant material testing to prevent outbreaks of disease. The planthopper Pentastiridius leporinus (Hempiptera: Cixiidae) is the main vector of two bacterial pathogens: the γ-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the stolbur phytoplasma 'Candidatus Phytoplasma solani'. These pathogens cause the disease syndrome basses richesses (SBR) in sugar beet (Beta vulgaris), which reduces the yields and sugar content. In 2022, potato (Solanum tuberosum) fields were found to be colonized by P. leporinus, and the transmission of Arsenophonus was confirmed, resulting in symptoms like wilting, yellow leaves, and rubbery tubers. We monitored both pathogens in Southwest Germany in 2022 and 2023. This revealed their widespread presence in potato tubers, although there were differences in regional prevalence. The broad prevalence of Arsenophonus was maintained in 2023, whereas the prevalence of stolbur increased in most locations. We confirmed that P. leporinus adults can transmit both pathogens to potatoes, but neither pathogen reduced the germination rate of tubers, and no plants showed abnormal growth after germination. Arsenophonus was not detected in germinated shoots, but 5.4% contained stolbur, emphasizing the need for plant material testing to maintain phytosanitary conditions. [ABSTRACT FROM AUTHOR]

Jujube witches’ broom (JWB) caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube. The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses. This study aims to identify and reveal the changes in the jujube GST gene family in response to phytoplasma infection. Here, 70 ZjGSTs were identified in the jujube genome and divided into 8 classes. Among them, the Tau-class, including 44 genes, was the largest. Phylogenetic analysis indicated that Tau-class genes were highly conserved among species, such as Arabidopsis, cotton, chickpea, and rice. Through chromosome location analysis, 37.1% of genes were clustered, and 8 of 9 gene clusters were composed of Tau class members. Through RT-PCR, qRT-PCR and enzyme activity detection, the results showed that the expression of half (20/40) of the tested ZjGSTs was inhibited by phytoplasma infection in field and tissue culture conditions, and GST activity was also significantly reduced. In the resistant and susceptible varieties under phytoplasma infection, ZjGSTU49-ZjGSTU54 in the cluster Ⅳ showed opposite expression patterns, which may be due to functional divergence during evolution. Some upregulated genes (ZjGSTU45, ZjGSTU49, ZjGSTU59, and ZjGSTU70) might be involved in the process of jujube against JWB. The yeast two-hybrid results showed that all 6 Tau-class proteins tested could form homodimers or heterodimers. Overall, the comprehensive analysis of the jujube GST gene family revealed that ZjGSTs responded actively to phytoplasma infection. Furthermore, some screened genes (ZjGSTU24, ZjGSTU49-52, ZjGSTU70, and ZjDHAR10) will contribute to further functional studies of jujube-phytoplasma interactions.

Phytoplasmas are intracellular pathogenic bacteria that infect a wide range of plant species, including agriculturally important crops and ornamental trees. However, our understanding of the relationship between symptom severity, disease progression, and phytoplasma concentration remains limited due to the inability to inoculate phytoplasmas mechanically into new plant hosts. The present study investigated phytoplasma titer dynamics and symptom development in periwinkle and tomato, both infected with the same potato purple top (PPT) phytoplasma strain using a small seedling grafting approach. Virescence, phyllody, and witches'-broom (WB) symptoms sequentially developed in periwinkle, while in tomato plants, big bud (BB, a form of phyllody), cauliflower-like inflorescence (CLI), and WB appeared in order. Results from quantitative polymerase chain reaction (qPCR) targeting the PPT phytoplasma's 16S rRNA gene revealed that in both host species, phytoplasma titers differed significantly at different infection stages. Notably, the highest phytoplasma concentration in periwinkles was observed in samples displaying phyllody symptoms, whereas in tomatoes, the titer peaked at the BB stage. Western blot analysis, utilizing an antibody specific to PPT phytoplasma, confirmed substantial phytoplasma presence in samples displaying phyllody and BB symptoms, consistent with the qPCR results. These findings challenge the conventional understanding that phytoplasma infection dynamics result in a higher titer at later stages, such as WB (excessive vegetative growth), rather than in the early stage, such as phyllody (abnormal reproductive growth). Furthermore, the PPT phytoplasma titer was markedly higher in periwinkles than in tomato plants, indicating differing susceptibilities between the hosts. This study reveals distinct host responses to PPT phytoplasma infection, providing valuable insights into phytoplasma titer dynamics and symptom development, with implications for the future management of agricultural disease. [ABSTRACT FROM AUTHOR]

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry. [ABSTRACT FROM AUTHOR]

Aster leafhopper (Hemiptera: Cicadellidae: Macrosteles quadrilineatus Forbes) is a polyphagous insect species that migrates into the upper Midwest of the United States and the Western Canadian Prairies. Populations of this insect are associated with the transmission of a plant pathogen (Candidatus Phytoplasma asteris, 16SrI) to several annual crops and perennial plant species. Previous studies suggest that aster leafhoppers can sometimes prefer less suitable hosts for their development and survival, yet it is unclear if this lower performance on certain plant species is associated with reduced or impaired probing behaviors due to characteristics of the plants. To characterize the probing behaviors of aster leafhoppers, direct current electropenetrography recordings of male and female adults on barley (Polaes: Poaceae: Hordeum vulgare L.) were combined with plant histology, allowing the identification of nine waveforms and their proposed biological meanings. For each waveform, the number of waveform events per insect (NWEI), the waveform duration per insect (WDI), the waveform duration per event per insect (WDEI), and the percentage of recording time were calculated and statistically compared between sexes. Male and female aster leafhoppers exhibited similar behavioral responses for most of these variables, except for the NWEI for waveforms associated with nonprobing activities and the pathway phase. In these cases, male aster leafhoppers exhibited a higher number of events than females. Comparison of the proposed waveforms in this study with previous work on other hemipteran species provided additional support to the interpretation of the biological activities associated with each waveform. [ABSTRACT FROM AUTHOR]

Pear decline (PD) phytoplasma populations were examined over one year in non-symptomatic pear trees with HW620 scions grafted onto three different rootstocks: OH×F87 (PD resistant), OH×F69 (PD susceptible), and Bartlett (PD susceptible). For all three rootstocks, populations were at a maximum during late summer for leaves and shoots, and reached their minimum in mid-winter for shoots and early spring for leaves. In contrast, roots exhibited maximum populations in mid-winter and minimum populations in mid-spring. For all tissue types, PD populations were consistently lowest in trees grafted onto OH×F87 rootstocks, intermediate in those on OH×F69, and highest on Bartlett rootstocks, demonstrating that the type of rootstock significantly impacts PD populations. While OH×F87 rootstocks had the lowest populations, they can still contain relatively high PD populations, particularly during periods with maximum populations. Future research could explore the development of even higher levels of PD resistance in pear rootstocks to reduce PD populations in both the rootstock and scion. [ABSTRACT FROM AUTHOR]

Phytoplasmas are a group of plant pathogens that reduce yield in various plants including many crops, shrubs, and tree species. They have been classified into various 16Sr groups or 'Candidatus Phytoplasma species' based primarily on the sequences of their 16S rRNA genes. However, other non-ribosomal sequences are often used to fine-tune their classifications. This research aimed to clarify the categorization of the sugarcane phytoplasmas that cause sugarcane white leaf and sugarcane grassy shoot symptoms in Thailand and determine whether they are different strains, based on non-16S ribosomal genes. Forty samples from plants exhibiting both sugarcane symptoms and from symptomless sugarcane were collected and PCR amplifications were done with primers for the 50S rRNA Ser and SecA genes moreover soil samples from non-symptom and symptom fields were collected for soil chemical properties and rhizospheric microorganism analysis. The phylogenetic tree results revealed that isolates displaying both symptom types grouped together based on all three sets of primers, including with isolates from other countries. Additionally, soil chemical properties and rhizospheric microorganism analysis results showed that soil chemical factors and rhizospheric microorganisms did not appear to correlate with the differences in symptoms. It is concluded that the two sugarcane symptoms are caused by the same Phytoplasma strain and that other, as yet unidentified factors, are responsible for this Phytoplasma giving different symptoms in different situations. This is the first evidence of soil chemical properties and rhizospheric microorganism not correlating with the diverse symptoms. As for further study, unidentified factors will be studied including quarantine methods to control the symptoms. [ABSTRACT FROM AUTHOR]

Phytoplasmas infect a wide variety of plants and can cause distinctive symptoms including the conversion of floral organs into leaf‐like organs, known as phyllody. Phyllody is induced by an effector protein family called phyllogens, which interact with floral MADS‐box transcription factors (MTFs) responsible for determining the identity of floral organs. The MTF/phyllogen complex then interacts with the proteasomal shuttle protein RADIATION SENSITIVE23 (RAD23), which facilitates delivery of the MTF/phyllogen complex to the host proteasome for MTF degradation. Previous studies have indicated that the MTF degradation specificity of phyllogens is determined by their ability to bind to MTFs. However, in the present study, we discovered a novel mechanism determining the degradation specificity through detailed functional analyses of a phyllogen homologue of rice yellow dwarf phytoplasma (PHYLRYD). PHYLRYD degraded a narrower range of floral MTFs than other phyllody‐inducing phyllogens, resulting in compromised phyllody phenotypes in plants. Interestingly, PHYLRYD was able to bind to some floral MTFs that PHYLRYD was unable to efficiently degrade. However, the complex of PHYLRYD and the non‐degradable MTF could not interact with RAD23. These results indicate that the MTF degradation specificity of PHYLRYD is correlated with the ability to form the MTF/PHYLRYD/RAD23 ternary complex, rather than the ability to bind to MTF. This study elucidated that phyllogen target specificity is regulated by both the MTF‐binding ability and RAD23 recruitment ability of the MTF/phyllogen complex. [ABSTRACT FROM AUTHOR]

'Candidatus Phytoplasma meliae' is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the 'Ca. P. meliae' strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique 'Ca. P. species' within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of 'Ca. P. meliae' ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for 'Ca. P. meliae' and others phytoplasmas for the 16SrXIII (MPV) group. [ABSTRACT FROM AUTHOR]

Grape hyacinths are popular perennial, flowering bulbous plants. In 2018, 2019, and 2020, some plants of three Muscari species showed symptoms similar to those associated with phytoplasma infection in commercial ornamental gardens in Hungary. Symptoms included virescence of flowers and yellowing of leaves. Symptomatic and asymptomatic Muscari plants were sampled at two locations to detect and identify the pathogens involved. Total DNA was extracted from the plants and used as a template in polymerase chain reaction assays to amplify 16S rRNA gene sequences and housekeeping genes (tuf, secY) with phytoplasma-specific primers. The resulting PCR products from symptomatic plants were cloned and sequenced bidirectionally. Homology searching of the obtained sequences against the GenBank database indicated the presence of 'Candidatus Phytoplasma asteris' in the three Muscari species. This is the first report worldwide of C. P. asteris phytoplasma infecting M. botryoides and M. comosum, and its first identification in M. armeniacum in Hungary.

The phytoplasma ‘Candidatus Phytoplasma pruni’, a causative agent of little cherry disease (LCD), has become an increasing problem for sweet cherry growers in Washington State, which is the largest producer of cherry fruit in the United States. The control of LCD currently relies on the identification and removal of infected trees, which has proven to be difficult because of the prolonged asymptomatic but still contagious state of the disease, and the lack of reliable and economical tests. Thus, the development of new approaches for early detection of LCD will be an important step in the successful control of this tree fruit disease. To identify potential microbial indicators of ‘Ca. P. pruni’ infection, we evaluated the bacterial and fungal communities in the roots of cherry trees from two different orchards that were (i) infected with ‘Ca. P. pruni’ and symptomatic; (ii) infected with ‘Ca. P. pruni’ but remained asymptomatic; and (iii) healthy, with non-‘Ca. P. pruni’-infected trees. We found significant variation in the microbiomes between the two cherry orchards, with the location being a stronger driving factor determining the fungal compared with the bacterial community. The fungal communities were less affected by the disease conditions compared with the bacterial microbiome. Overall, this study demonstrates the feasibility of the microbiome approach for the early detection of LCD caused by ‘Ca. P. pruni’ but also demonstrates that more orchards need to be sampled because location was a stronger contributor to the microbiome of cherry tree roots than disease condition.

Cacopsylla mali, C. melanoneura, C. picta, C. pruni, C. pyri, and C. pyrisuga belong among important pests of fruit trees. They harm trees by sucking and transmitting pathogenic bacteria of the ‘Candidatus Phytoplasma’, 16SrX group, which leads to declining and death of fruit trees. Protection against these pests depends on their early reliable identification and monitoring in fruit nurseries and orchards. In general, the determination of adult psyllids is based on morphological criteria, however, it is difficult or even impossible to perform determination using eggs and nymphs. In this study, a recently developed real-time PCR method validated for the determination of adult psyllids, was applied for the identification of other developmental stages. A total, 46 samples of eggs and nymph instars N1 to N5 were successfully determined, indicating the PCR identification system can be reliably used also for these types of specimens. Specific PCRs detected three species – C. melanoneura, C. pyri a C. pruni which were also confirmed by sequencing of the cytochrome c oxidase subunit I (COI) gene. Thus, this innovative molecular method can be used for a quick and routine determination of selected Cacopsylla species infecting fruit trees in all developmental stages.

Cucumber plants (Cucumis sativus) displaying symptoms of phyllody, sterility, and flower virescence were observed in fields located in the Dianbai district of Guangdong province, China. Total DNA was extracted from the symptomatic plants; this was followed by molecular detection using a set of universal phytoplasma primer pairs, R16mF2/mR1, P1/P7, and SecYF1(II)/SecYR1(II). This resulted in the PCR amplification of products corresponding to expected sizes of 1.4 kb, 1.8 kb and 1.7 kb, respectively. The 16S rDNA sequence obtained exhibited 100% similarity with the eggplant phyllody phytoplasma, the ‘Cleome rutidosperma’ witches’ broom phytoplasma, and the ‘Desmodium ovalifolium’ witches’ broom phytoplasma strain DeOWB, all of which belong to the 16SrII group. Phylogenetic analysis, based on the 16S rDNA gene and SecY gene sequences, confirmed the close affiliation of the detected phytoplasma isolate, tentatively designated as cucumber phyllody phytoplasma (CuPh) China isolate, with the 16SrII-A subgroup. Additionally, virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA sequence revealed a pattern that was identical to that of the 16SrII-A subgroup. This is the first report of cucumber phyllody phytoplasma in China.

The bacterial genus Wolbachia induces reproductive abnormalities in its insect host, including cytoplasmic incompatibility (CI), which causes embryonic death in the crossing of infected males and uninfected females. Hence, Wolbachia-based strategies are employed to control insect pests. However, Wolbachia does not naturally infect Matsumuratettix hiroglyphicus (Matsumura), the main vector of the phytoplasma causing the sugarcane white leaf (SCWL) disease. In this study, the wYfla Wolbachia strain, which induces strong CI in its original host, was microinjected into nymphs of M. hiroglyphicus. Molecular detection revealed that Wolbachia was successfully transinfected into the recipient host, with an infection frequency of 55–80% in up to eight generations after transinfection. Wolbachia exhibited no significant detrimental effects on the developmental time of the immature stages, adult emergences, and female longevity, whereas the lifespan of transinfected males was decreased. Reciprocal crossing revealed that Wolbachia infection did not affect the number of eggs laid per female. However, the hatching rate produced by the pairs between the transinfected males and naturally uninfected females significantly decreased. The evidence of Wolbachia transmitted through the generations tested and partial CI occurrence in transinfected M. hiroglyphicus highlights the possibility of the future development of Wolbachia-based strategies for controlling the vector of SCWL.

Introduction: The adhesion of flavescence doreée phytoplasma to the midgut epithelium cells of their insect vectors is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. In order to identify the insect receptor for VmpA, we identified Euscelidius variegatus cell proteins interacting with recombinant VmpA-His6. Methods: The E. variegatus proteins were identified by mass spectrometry analysis of VmpA-E. variegatus protein complexes formed upon in vitro interaction assays. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of E. variegatus cells to bind VmpA-coated fluorescent beads. Results and discussion: There were 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains selected. The decrease of expression of an unknown transmembrane protein with leucine-rich repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA beads to E. variegatus cells. The uk1_LRR was more expressed in digestive tubes than salivary glands of E. variegatus. The protein uk1_LRR could be implicated in the binding with VmpA in the early stages of insect infection following phytoplasmas ingestion [ABSTRACT FROM AUTHOR]

Apple proliferation, caused by 'Candidatus Phytoplasma mali', is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of 'Golden Delicious'. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars. [ABSTRACT FROM AUTHOR]

Phytoplasmas are phloem-limited plant pathogens, such as sugarcane white leaf (SCWL) phytoplasma, which are responsible for heavy economic losses to the sugarcane industry. Characterization of phytoplasmas has been limited because they cannot be cultured in vitro. However, with the advent of genome sequencing, dierent aspects of phytoplasmas are being investigated. In this study, we developed a DNA enrichment method for sugarcane white leaf (SCWL) phytoplasma, evaluated the eect of DNA enrichment via Illumina sequencing technologies, and utilized Illumina and Nanopore sequencing technologies to obtain the complete genome sequence of the “Candidatus Phytoplasma sacchari” isolate SCWL1 that is associated with sugarcane white leaf in China. Illumina sequencing analysis elucidated that only 1.21% of the sequencing reads from total leaf DNA were mapped to the SCWL1 genome, whereas 40.97% of the sequencing reads from the enriched DNA were mapped to the SCWL1 genome. The genome of isolate SCWL1 consists of a 538,951 bp and 2976 bp long circular chromosome and plasmid, respectively. We identified 459 protein-encoding genes, 2 complete 5S-23S-16S rRNA gene operons, 27 tRNA genes, and an incomplete potential mobile unit (PMU) in the circular chromosome. Phylogenetic analyses and average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values based on the sequenced genome revealed that SCWL phytoplasma and sugarcane grassy shoot (SCGS) phytoplasma belonged to the same phytoplasma species. This study provides a genomic DNA enrichment method for phytoplasma sequencing. Moreover, we report the first complete genome of a “Ca. Phytoplasma sacchari” isolate, thus contributing to future studies on the evolutionary relationships and pathogenic mechanisms of “Ca. Phytoplasma sacchari” isolates. [ABSTRACT FROM AUTHOR]

Grape hyacinths are popular perennial, flowering bulbous plants. In 2018, 2019, and 2020, some plants of three Muscari species showed symptoms similar to those associated with phytoplasma infection in commercial ornamental gardens in Hungary. Symptoms included virescence of flowers and yellowing of leaves. Symptomatic and asymptomatic Muscari plants were sampled at two locations to detect and identify the pathogens involved. Total DNA was extracted from the plants and used as a template in polymerase chain reaction assays to amplify 16S rRNA gene sequences and housekeeping genes (tuf, secY) with phytoplasma-specific primers. The resulting PCR products from symptomatic plants were cloned and sequenced bidirectionally. Homology searching of the obtained sequences against the GenBank database indicated the presence of 'Candidatus Phytoplasma asteris' in the three Muscari species. This is the first report worldwide of C. P. asteris phytoplasma infecting M. botryoides and M. comosum, and its first identification in M. armeniacum in Hungary. [ABSTRACT FROM AUTHOR]

Plant pathogens that are transmitted by insect vectors cause considerable damage to crops when pests or pathogens are not detected early in the season and populations are not controlled. Knowledge of pathogen prevalence in insect pest populations can aid growers in their insect pest management decisions but requires the timely dissemination of results. This process requires that specimen capture, identification, nucleic acid extraction, and molecular detection of a pathogen(s) occur alongside a platform for sharing results. The potato psyllid (Bactericera cockerelli , Sulc; Hemiptera: Triozidae) and beet leafhopper (Circulifer tenellus , Baker; Hemiptera: Cicadellidae) transmit pathogens to potato and other vegetable or seed crops each season in the northwestern United States. While the potato psyllid has been tested for pathogen occurrence for the past decade, testing of the beet leafhopper is a new endeavor and substantially increases the specimen number that must be tested by our laboratories each season. To aid in the rapid processing of individual insect specimens, we optimized and validated a new high-throughput 96-well plate nucleic acid extraction method for use in place of a standard 1.5-ml single-tube extraction method. Processing efficiency, in terms of total specimens processed over a 2-day period, improved 2.5-fold, and the cost associated with processing a single sample was nearly cut in half with this newly developed plate nucleic acid extraction method. Overall, this method has proven to be an excellent tool for the rapid testing of large numbers of small, individual insect vectors to enable timely dissemination of data on pathogen prevalence to growers. [ABSTRACT FROM AUTHOR]

Camptotheca acuminata Witches'-broom disease (CaWB) is the most destructive disease affecting C. acuminata in China. Previous studies on CaWB have failed to clarify the incidence pattern in C. acuminata after infection with phytoplasma. The time interval between phytoplasma infection of C. acuminata and the onset of Witches'-broom symptoms in C. acuminata was very long. C. acuminata inoculated with CaWB showed leaf margin scorching symptoms at 4 weeks in inoculated leaves. At 16 weeks after infection (WAI), old leaves were shed, while new leaves showed a mild leaf margin scorch; at 28 WAI, typical symptoms appeared. Transcriptomic and metabolomic analyses of the three sampling periods revealed 194 differentially expressed genes, mainly enriched in MAPK signaling, plant–pathogen interaction, phenylpropanoid biosynthesis, starch and phenylpropanoid biosynthesis, and phenylpropanoid biosynthesis pathways. The expression of calcium-dependent protein kinase (CDPK), β Ketoacyl-CoA Synthase1/10 (KCS1/10), and WRKY22/29 genes in the plant–pathogen interaction pathway significantly increased, indicating that they may be key genes in the CaWB phytoplasma-mediated maintenance of ROS homeostasis. Moreover, isochlorogenic acid B, atractylenolide II, and 3-methoxybenzoic acid were found, which might serve as signaling or functional substances in the defense response. Our results provide novel insights into the pathogenesis of CaWB and the defense response of C. acuminata under the influence of phytoplasma. Additionally, we identified potential candidate genes related to the defense response of C. acuminata, laying the foundation for further research. [ABSTRACT FROM AUTHOR]

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