Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe.

In Europe, two fastidious phloem-limited pathogens, 'Candidatus Phytoplasma solani' (16SrXII-A) and 'Candidatus Arsenophonus phytopathogenicus', are associated with rubbery taproot disease (RTD) and syndrome basses richesses (SBR) of sugar beet, respectively. Both diseases can significantly reduce yield, especially when accompanied by root rot fungi. This study investigates the presence, geographic distribution and genetic traits of fastidious pathogens and the accompanying fungus, Macrophomina phaseolina, found on sugar beet across four geographically separated plains spanning seven countries in Central Europe. The survey revealed variable incidences of symptoms linked to these fastidious pathogens in the Pannonian and Wallachian Plains, sporadic occurrence in the North European Plain, and no symptomatic sugar beet in the Bohemian Plain. Molecular analyses unveiled the occurrence of both 'Ca. P. solani' and 'Ca. A. phytopathogenicus' throughout Central Europe, with a predominance of the phytoplasma. These fastidious pathogens were detected in all six countries surveyed within the Pannonian and Wallachian Plains, with only a limited presence of various phytoplasmas was found in the North European Plain, while no fastidious pathogens were detected in Bohemia, aligning with observed symptoms. While 16S rDNA sequences of 'Ca. P. solani' remained highly conserved, multi-locus characterization of two more variable loci (tuf and stamp) unveiled distinct variability patterns across the plains. Notably, the surprising lack of variability of tuf and stamp loci within Central Europe, particularly the Pannonian Plain, contrasted their high variability in Eastern and Western Europe, corresponding to epidemic and sporadic occurrence, respectively. The current study provides valuable insights into the genetic dynamics of 'Ca. P. solani' in Central Europe, and novel findings of the presence of 'Ca. A. phytopathogenicus' in five countries (Slovakia, Czech Republic, Austria, Serbia, and Romania) and M. phaseolina in sugar beet in Slovakia. These findings emphasize the need for further investigation of vector-pathogen(s)-plant host interactions and ecological drivers of disease outbreaks.

Protocol for the Acclimatization of Coconut Vitro-Plants Under Ex Vitro Conditions.

The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.

Comprehensive analysis of little leaf disease incidence and resistance in eggplant.

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.

The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses.

BACKGROUND: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear. RESULTS: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity. CONCLUSIONS: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

Detection and identification of Bogia coconut syndrome phytoplasma from seed-associated tissues and seedlings of coconut (Cocos nucifera) and betel nut (Areca catechu).

Evidence for seed transmission of phytoplasmas has grown in several pathosystems including coconut (Cocos nucifera). Bogia coconut syndrome (BCS) is a disease associated with the lethal yellowing syndrome associated with the presence of 'Candidatus Phytoplasma noviguineense' that affects coconut, betel nut (Areca catechu) and bananas (Musa spp.) in Papua New Guinea. Coconut and betel nut drupes were sampled from BCS-infected areas in Papua New Guinea, dissected, the extracted nucleic acid was used in polymerase chain reaction (PCR), and loop mediated isothermal amplification (LAMP) used to check for presence of phytoplasma DNA. In a second study, drupes of both plant species were collected from multiple field sites and grown in insect-proof cages. Leaf samples taken at 6 months were also tested with PCR and LAMP. The studies of dissected coconut drupes detected phytoplasma DNA in several tissues including the embryo. Drupes from betel nut tested negative. Among the seedlings, evidence of possible seed transmission was found in both plant species. The results demonstrate the presence of 'Ca. P. noviguineense' in coconut drupes and seedlings, and in seedlings of betel nut; factors that need to be considered in ongoing management and containment efforts.

A phytoplasma effector destabilizes chloroplastic glutamine synthetase inducing chlorotic leaves that attract leafhopper vectors.

Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.

'Candidatus Phytoplasma mali' SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves.

BACKGROUND: 'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11CaPm which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11CaPm. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11CaPm and changes in the transcriptome were recorded until 5 days post infiltration. RESULTS: The RNA-seq analysis revealed that presence of SAP11CaPm in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced. CONCLUSIONS: The results indicate that early SAP11CaPm expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.

Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

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.

The 'Candidatus phytoplasma ziziphi' effectors SJP1 and SJP2 destabilise the bifunctional regulator ZjTCP7 to modulate floral transition and shoot branching.

Phytoplasmic SAP11 effectors alter host plant architecture and flowering time. However, the exact mechanisms have yet to be elucidated. Two SAP11-like effectors, SJP1 and SJP2, from 'Candidatus Phytoplasma ziziphi' induce shoot branching proliferation. Here, the transcription factor ZjTCP7 was identified as a central target of these two effectors to regulate floral transition and shoot branching. Ectopic expression of ZjTCP7 resulted in enhanced bolting and earlier flowering than did the control. Interaction and expression assays demonstrated that ZjTCP7 interacted with the ZjFT-ZjFD module, thereby enhancing the ability of these genes to directly bind to the ZjAP1 promoter. The effectors SJP1 and SJP2 unravelled the florigen activation complex by specifically destabilising ZjTCP7 and ZjFD to delay floral initiation. Moreover, the shoot branching of the ZjTCP7-SRDX transgenic Arabidopsis lines were comparable to those of the SJP1/2 lines, suggesting the involvement of ZjTCP7 in the regulation of shoot branching. ZjTCP7 interacted with the branching repressor ZjBRC1 to enhance suppression of the auxin efflux carrier ZjPIN3 expression. ZjTCP7 also directly bound to and upregulated the auxin biosynthesis gene ZjYUCCA2, thereby promoting auxin accumulation. Our findings confirm that ZjTCP7 serves as a bifunctional regulator destabilised by the effectors SJP1 and SJP2 to modulate plant development.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

The Role of Plant Defense Signaling Pathways in Phytoplasma-Infected and Uninfected Aster Leafhoppers' Oviposition, Development, and Settling Behavior.

In plant-microbe-insect systems, plant-mediated responses involve the regulation and interactions of plant defense signaling pathways of phytohormones jasmonic acid (JA), ethylene (ET), and salicylic acid (SA). Phytoplasma subgroup 16SrI is the causal agent of Aster Yellows (AY) disease and is primarily transmitted by populations of aster leafhoppers (Macrosteles quadrilineatus Forbes). Aster Yellows infection in plants is associated with the downregulation of the JA pathway and increased leafhopper oviposition. The extent to which the presence of intact phytohormone-mediated defensive pathways regulates aster leafhopper behavioral responses, such as oviposition or settling preferences, remains unknown. We conducted no-choice and two-choice bioassays using a selection of Arabidopsis thaliana lines that vary in their defense pathways and repeated the experiments using AY-infected aster leafhoppers to evaluate possible differences associated with phytoplasma infection. While nymphal development was similar among the different lines and groups of AY-uninfected and AY-infected insects, the number of offspring and individual female egg load of AY-uninfected and AY-infected insects differed in lines with mutated components of the JA and SA signaling pathways. In most cases, AY-uninfected insects preferred to settle on wild-type (WT) plants over mutant lines; no clear pattern was observed in the settling preference of AY-infected insects. These findings support previous observations in other plant pathosystems and suggest that plant signaling pathways and infection with a plant pathogen can affect insect behavioral responses in more than one manner. Potential differences with previous work on AY could be related to the specific subgroup of phytoplasma involved in each case.

Characterization and correlation of the probing behaviors of Macrosteles quadrilineatus (Hemiptera: Cicadellidae) with electropenetrography (EPG) waveforms.

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.

Insect Vector and Reservoir Plant of 'Fragaria × ananassa' Phyllody Phytoplasma (16SrXIII-F) in Central Region of Chile.

Strawberry phyllody has emerged as a prevalent disease affecting Chilean strawberry in recent years. The causal pathogen, 'Fragaria × ananassa' phyllody phytoplasma (StrPh), is categorized within the 16S ribosomal group XIII that is exclusively found in the Americas. In the context of economically significant crops, hemipteran insect vectors and alternative host plants play a pivotal role in their natural dissemination. This study comprehensively examined the key epidemiological facets of StrPh in the central region of Chile: the insect vector and alternative hosts. Through field surveys, we identified an abundance of an insect species, Cixiosoma sp., in an StrPh-infected strawberry field and confirmed its role as a vector of this phytoplasma through subsequent transmission assays. Moreover, we found a spontaneous weed species, Galega officinalis, to be infected with StrPh, raising the possibility of it being a potential alternative host plant for this phytoplasma. StrPh was also detected in cold-stored strawberry runners purchased from a nursery that supplies the local strawberry cultivation, suggesting a potential source of this phytoplasma in Chile. Collectively, these findings provide a significant epidemiological source of StrPh dissemination in central Chile.

The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing the transcription factor ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

Genome-Wide Identification of the Paulownia fortunei Aux/IAA Gene Family and Its Response to Witches' Broom Caused by Phytoplasma.

The typical symptom of Paulownia witches' broom (PaWB), caused by phytoplasma infection, is excessive branching, which is mainly triggered by auxin metabolism disorder. Aux/IAA is the early auxin-responsive gene that participates in regulating plant morphogenesis such as apical dominance, stem elongation, lateral branch development, and lateral root formation. However, no studies have investigated the response of the Aux/IAA gene family to phytoplasma infection in Paulownia fortunei. In this study, a total of 62 Aux/IAA genes were found in the genome. Phylogenetic analysis showed that PfAux/IAA genes could be divided into eight subgroups, which were formed by tandem duplication and fragment replication. Most of them had a simple gene structure, and several members lacked one or two conserved domains. By combining the expression of PfAux/IAA genes under phytoplasma stress and SA-treated phytoplasma-infected seedlings, we found that PfAux/IAA13/33/45 may play a vital role in the occurrence of PaWB. Functional analysis based on homologous relationships showed a strong correlation between PfAux/IAA45 and branching. Protein-protein interaction prediction showed that PfARF might be the binding partner of PfAux/IAA, and the yeast two-hybrid assay and bimolecular fluorescent complementary assay confirmed the interaction of PfAux/IAA45 and PfARF13. This study provides a theoretical basis for further understanding the function of the PfAux/IAA gene family and exploring the regulatory mechanism of branching symptoms caused by PaWB.

Comprehensive Analysis of the GRAS Gene Family in Paulownia fortunei and the Response of DELLA Proteins to Paulownia Witches' Broom.

The GRAS (GAI\RGA\SCL) gene family encodes plant-specific transcription factors that play crucial roles in plant growth and development, stress tolerance, and hormone network regulation. Plant dwarfing symptom is mainly regulated by DELLA proteins of the GRAS gene subfamily. In this study, the association between the GRAS gene family and Paulownia witches' broom (PaWB) was investigated. A total of 79 PfGRAS genes were identified using bioinformatics methods and categorized into 11 groups based on amino acid sequences. Tandem duplication and fragment duplication were found to be the main modes of amplification of the PfGRAS gene family. Gene structure analysis showed that more than 72.1% of the PfGRASs had no introns. The genes PfGRAS12/18/58 also contained unique DELLA structural domains; only PfGRAS12, which showed significant response to PaWB phytoplasma infection in stems, showed significant tissue specificity and responded to gibberellin (GA3) in PaWB-infected plants. We found that the internodes were significantly elongated under 100 µmol·L-1 GA3 treatment for 30 days. The subcellular localization analysis indicated that PfGRAS12 is located in the nucleus and cell membrane. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays confirmed that PfGRAS12 interacted with PfJAZ3 in the nucleus. Our results will lay a foundation for further research on the functions of the PfGRAS gene family and for genetic improvement and breeding of PaWB-resistant trees.

Phytoplasma: A plant pathogen that cannot be ignored in agricultural production-Research progress and outlook.

Phytoplasmas are phloem-restricted plant-pathogenic bacteria transmitted by insects. They cause diseases in a wide range of host plants, resulting in significant economic and ecological losses worldwide. Research on phytoplasmas has a long history, with significant progress being made in the past 30 years. Notably, with the rapid development of phytoplasma research, scientists have identified the primary agents involved in phytoplasma transmission, established classification and detection systems for phytoplasmas, and 243 genomes have been sequenced and assembled completely or to draft quality. Multiple possible phytoplasma effectors have been investigated, elucidating the molecular mechanisms by which phytoplasmas manipulate their hosts. This review summarizes recent advances in phytoplasma research, including identification techniques, host range studies, whole- or draft-genome sequencing, effector pathogenesis and disease control methods. Additionally, future research directions in the field of phytoplasma research are discussed.

Molecular Identification and Characterization of Novel Taxonomic Subgroups and New Host Plants in 16SrI and 16SrII Group Phytoplasmas and Their Evolutionary Diversity on Hainan Island, China.

Phytoplasmas are a group of plant prokaryotic pathogens distributed worldwide. To comprehensively reveal the diversity of the pathogens and the diseases they cause on Hainan, a tropical island with abundant biodiversity in China, a survey of phytoplasmal diseases was performed from 2009 to 2022. Herein, molecular identification and genetic analysis were conducted based on the conserved genes of phytoplasmas. The results indicated that phytoplasmas could be detected in 138 samples from 18 host plants among 215 samples suspected to be infected by the pathogens. The phytoplasma strains from 27 diseased samples of 4 host plants belonged to the 16SrI group and the strains from 111 samples of 14 hosts belonged to the 16SrII group. Among them, 12 plants, including important tropical cash crops such as Phoenix dactylifera, cassava, sugarcane, and Piper nigrum, were first identified as hosts of phytoplasmas on Hainan Island. Based on BLAST and iPhyClassifier analyses, seven novel 16Sr subgroups were proposed to describe the relevant phytoplasma strains, comprising the 16SrI-AP, 16SrI-AQ, and 16SrI-AR subgroups within the 16SrI group and the 16SrII-Y, 16SrII-Z, 16SrII-AB, and 16SrII-AC subgroups within the 16SrII group. Genetic variation and phylogenetic analysis indicated that the phytoplasma strains identified in this study and those reported previously on Hainan Island mainly belong to four 16Sr groups (including I, II, V, and XXXII) and could infect 44 host plants, among which the 16SrI and 16SrII groups were the prevalent 16Sr groups associated with 43 host plant species. The diversity of host plants infected by the phytoplasmas made it difficult to monitor and control their related diseases. Therefore, strengthening inspection and quarantine during the introduction and transit of the related phytoplasmal host crops would effectively curb the spread and prevalence of the phytoplasmas and their related lethal diseases.

Jujube Witches' Broom Phytoplasma Effector Zaofeng3, a Homologous Effector of SAP54, Induces Abnormal Floral Organ Development and Shoot Proliferation.

Plant-pathogenic phytoplasmas secrete specific virulence proteins into a host plant to modulate plant function for their own benefit. Identification of phytoplasmal effectors is a key step toward clarifying the pathogenic mechanisms of phytoplasma. In this study, Zaofeng3, also known as secreted jujube witches' broom phytoplasma protein 3 (SJP3), was a homologous effector of SAP54 and induced a variety of abnormal phenotypes, such as phyllody, malformed floral organs, witches' broom, and dwarfism in Arabidopsis thaliana. Zaofeng3 can also induce small leaves, dwarfism, and witches' broom in Ziziphus jujuba. Further experiments showed that the three complete α-helix domains predicted in Zaofeng3 were essential for induction of disease symptoms in jujube. Yeast two-hybrid library screening showed that Zaofeng3 mainly interacts with proteins involved in flower morphogenesis and shoot proliferation. Bimolecular fluorescence complementation assays confirmed that Zaofeng3 interacted with these proteins in the whole cell. Overexpression of zaofeng3 in jujube shoot significantly altered the expression patterns of ZjMADS19, ZjMADS47, ZjMADS48, ZjMADS77, and ZjTCP7, suggesting that overexpressing zaofeng3 might induce floral organ malformation and witches' broom by altering the expression of the transcriptional factors involved in jujube morphogenesis.