Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana.

Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana's pathogenic mechanisms.

Diversity and composition of the bacterial communities associated with the Australian spittlebugs Bathyllus albicinctus and Philagra parva (Hemiptera: Aphrophoridae).

Spittlebugs and froghoppers (Hemiptera: Cercopoidea) are insects feeding on xylem, which potentially can cause significant economic damage worldwide by transmitting plant pathogenic bacteria such as Xylella fastidiosa. Australia and New Zealand are currently free from X. fastidiosa, but they are home to at least 45 native spittlebug species. Among these, the Australian natives Bathyllus albicinctus (Erichson, 1842) and Philagra parva (Donovan, 1805) are particularly widespread and can be found across southern and eastern Australia, with B. albicinctus also in New Zealand. The potential that both species might be capable of vectoring Xylella fastidiosa poses a substantial biosecurity risk if the bacterium were to invade these regions. In this study, we examined 87 spittlebug nymphs collected across 12 different host plant species, in five locations in Victoria, Australia. Our objective was to explore the factors influencing bacterial communities within and between these widespread spittlebug species, considering geographic location, insect phylogenetics, and host plant associations. We employed COI barcoding to assess insect genetic variation and 16S high throughput sequencing (HTS) metabarcoding to analyse bacterial microbiome diversity across various host plants. Our findings revealed minimal genetic divergence among spittlebug individuals in the same species, highlighting conspecificity despite conspicuous morphological divergences. On the other hand, we recorded significant variation in bacterial communities harboured by Bathyllus albicinctus nymphs feeding on different plants, even when these were collected within close proximity to each other. Therefore, host plant association appeared to shape the bacterial communities of spittlebugs more than insect genetic divergence or geographical location. These diverse bacterial communities could potentially facilitate transmission of plant pathogenic bacteria, underscoring the risk of widespread transmission among numerous plant hosts through insect-plant interactions. This study emphasizes the critical need to understand these complex interactions, particularly in the context of biosecurity.

Innovative strategies for characterizing and managing huanglongbing in citrus.

Huanglongbing is a severe citrus disease that causes significant tree and crop losses worldwide. It is caused by three Candidatus liberibacter species and spread by psyllids and infected budwood. Various methods have been used to diagnose and understand HLB, including recent advances in molecular and biochemical assays that explore the pathogen's mode of action and its impact on the host plant. Characterization is essential for developing sustainable HLB management strategies. Nanotechnology, particularly nano sensors and metal nanoparticles, shows potential for precise disease diagnosis and control. Additionally, antibiotics, nanomaterials, and genetic engineering techniques like transgenesis offer promising avenues for mitigating HLB. These diverse approaches, from conventional to cutting-edge, contribute to developing integrated HLB management strategies for sustainable citrus cultivation. The review highlights the significant advancements in conventional and advanced molecular and biochemical characterization of HLB, aiding in early detection and understanding of the infection mechanism. It emphasizes the multidimensional efforts required to characterize disease and devise innovative management strategies. As the citrus industry faces unprecedented challenges, exploring new frontiers in HLB research provides hope for sustainable solutions and a resilient future for global citrus cultivation.

Highly Reduced Complementary Genomes of Dual Bacterial Symbionts in the Mulberry Psyllid Anomoneura mori.

The genomes of obligately host-restricted bacteria suffer from accumulating mildly deleterious mutations, resulting in marked size reductions. Psyllids (Hemiptera) are phloem sap-sucking insects with a specialized organ called the bacteriome, which typically harbors two vertically transmitted bacterial symbionts: the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria) and a secondary symbiont that is phylogenetically diverse among psyllid lineages. The genomes of several Carsonella lineages were revealed to be markedly reduced (158-174| |kb), AT-rich (14.0-17.9% GC), and structurally conserved with similar gene inventories devoted to synthesizing essential amino acids that are scarce in the phloem sap. However, limited genomic information is currently available on secondary symbionts. Therefore, the present study investigated the genomes of the bacteriome-associated dual symbionts, Secondary_AM (Gammaproteobacteria) and Carsonella_AM, in the mulberry psyllid Anomoneura mori (Psyllidae). The results obtained revealed that the Secondary_AM genome is as small and AT-rich (229,822 bp, 17.3% GC) as those of Carsonella lineages, including Carsonella_AM (169,120 bp, 16.2% GC), implying that Secondary_AM is an evolutionarily ancient obligate mutualist, as is Carsonella. Phylogenomic ana-lyses showed that Secondary_AM is sister to "Candidatus Psyllophila symbiotica" of Cacopsylla spp. (Psyllidae), the genomes of which were recently reported (221-237| |kb, 17.3-18.6% GC). The Secondary_AM and Psyllophila genomes showed highly conserved synteny, sharing all genes for complementing the incomplete tryptophan biosynthetic pathway of Carsonella and those for synthesizing B vitamins. However, sulfur assimilation and carotenoid-synthesizing genes were only retained in Secondary_AM and Psyllophila, respectively, indicating ongoing gene silencing. Average nucleotide identity, gene ortholog similarity, genome-wide synteny, and substitution rates suggest that the Secondary_AM/Psyllophila genomes are more labile than Carsonella genomes.

Gene expression profiling of Cacopsylla pyricola (Hemiptera: Psyllidae) infected with Ca. Phytoplasma pyri (Acholeplasmatales: Acholeplasmataceae) reveals candidate effectors and mechanisms of infection.

Phytoplasmas can negatively or positively alter vector host fitness. "Candidatus Phytoplasma pyri," is the causal agent of pear decline in commercial pear (Pyrus communis L.; Rosales: Rosaceae) and peach yellow leafroll in peach [Prunus persica (L.); Rosaceae]. This plant pathogen is transmitted by several species of pear psyllids (Cacopsylla spp. Hemiptera: Psyllidae). We sought to explore the relationship between the pear decline phytoplasma and its US vector, Cacopsylla pyricola (Förster), at the molecular genetic level through transcriptomic analysis using RNA-sequencing methodology. We also focused on phytoplasma and insect effectors, which are secreted proteins that can modulate interactions within a pathosystem. In this study, we identified 30 differentially expressed genes, 14 candidate insect effector genes, and 8 Ca. Phytoplasma pyri candidate effectors. Two strains of Ca. Phytoplasma pyri were identified based on immunodominant membrane protein sequence analysis from C. pyricola collected in the Pacific Northwest agricultural region. Here, we present a first genetic look at the pear decline pathosystem and report gene candidates for further exploration of infection mechanisms and potential tools for integrated pest management.

Rapid Loss of Nutritional Symbionts in an Endemic Hawaiian Herbivore Radiation Is Associated with Plant Galling Habit.

Insect herbivores frequently cospeciate with symbionts that enable them to survive on nutritionally unbalanced diets. While ancient symbiont gain and loss events have been pivotal for insect diversification and feeding niche specialization, evidence of recent events is scarce. We examine the recent loss of nutritional symbionts (in as little as 1 MY) in sap-feeding Pariaconus, an endemic Hawaiian insect genus that has undergone adaptive radiation, evolving various galling and free-living ecologies on a single host-plant species, Metrosideros polymorpha within the last ∼5 MY. Using 16S rRNA sequencing, we investigated the bacterial microbiomes of 19 Pariaconus species and identified distinct symbiont profiles associated with specific host-plant ecologies. Phylogenetic analyses and metagenomic reconstructions revealed significant differences in microbial diversity and functions among psyllids with different host-plant ecologies. Within a few millions of years, Pariaconus species convergently evolved the closed-gall habit twice. This shift to enclosed galls coincided with the loss of the Morganella-like symbiont that provides the essential amino acid arginine to free-living and open-gall sister species. After the Pariaconus lineage left Kauai and colonized younger islands, both open- and closed-gall species lost the Dickeya-like symbiont. This symbiont is crucial for synthesizing essential amino acids (phenylalanine, tyrosine, and lysine) as well as B vitamins in free-living species. The recurrent loss of these symbionts in galling species reinforces evidence that galls are nutrient sinks and, combined with the rapidity of the evolutionary timeline, highlights the dynamic role of insect-symbiont relationships during the diversification of feeding ecologies. We propose new Candidatus names for the novel Morganella-like and Dickeya-like symbionts.

The symbiont Acinetobacter baumannii enhances the insect host resistance to entomopathogenic fungus Metarhizium anisopliae.

Major symbiotic organisms have evolved to establish beneficial relationships with hosts. However, understanding the interactions between symbionts and insect hosts, particularly for their roles in defense against pathogens, is still limited. In a previous study, we proposed that the fungus Metarhizium anisopliae can infect the brown planthopper Nilaparvata lugens, a harmful pest for rice crops. To expand on this, we investigated changes in N. lugens' intestinal commensal community after M. anisopliae infection and identified key gut microbiotas involved. Our results showed significant alterations in gut microbiota abundance and composition at different time points following infection with M. anisopliae. Notably, certain symbionts, like Acinetobacter baumannii, exhibited significant variations in response to the fungal infection. The decrease in these symbionts had a considerable impact on the insect host's survival. Interestingly, reintroducing A. baumannii enhanced the host's resistance to M. anisopliae, emphasizing its role in pathogen defense. Additionally, A. baumannii stimulated host immune responses, as evidenced by increased expression of immune genes after reintroduction. Overall, our findings highlight the significance of preserving a stable gut microbial community for the survival of insects. In specific conditions, the symbiotic microorganism A. baumannii can enhance the host's ability to resist entomopathogenic pathogens through immune regulation.

Rich diversity of RNA viruses in the biological control agent, Orius laevigatus.

Orius laevigatus (Hemiptera, Anthocoridae) is a generalist predator extensively used for the biocontrol of diverse agricultural pests. Previous studies on O. laevigatus have focused on the improvement of insect genetic traits, but little is known about its association with microbes, especially viruses that may influence its production and efficacy. More than 280 RNA viruses have been described in other Hemiptera insects, in line with the continuous discovery of insect-specific viruses (ISVs) boosted by next-generation sequencing. In this study, we characterized the repertoire of RNA viruses associated with O. laevigatus. Its virome comprises 27 RNA viruses, classified within fourteen viral families, of which twenty-three viruses are specific to O. laevigatus and four are likely associated with fungal microbiota. The analysis of viral abundance in five O. laevigatus populations confirmed the presence of simultaneous viral infections and highlighted the ubiquitous presence and high abundance of one solinvivirus and three totiviruses. Moreover, we identified 24 non-retroviral endogenous viral elements (nrEVEs) in the genome of O. laevigatus, suggesting a long-term relationship between the host and its virome. Although no symptoms were described in the insect populations under study, the high diversity of viral species and the high abundance of certain RNA viruses identified indicate that RNA viruses may be significant for the applicability and efficacy of O. laevigatus in biocontrol programs.

Mass production of Isaria fumosorosea pfu5 for community approach management of rugose spiraling whitefly in oil palm.

The management of an alien Rugose spiraling whitefly (RSW) on oil palm using a native entomopathogenic fungus, Isaria fumosorosea pfu5 necessitated community approach for pest management. Moreover, coverage of huge leaf biomass warrants massive multiplication of biocontrol agent. In this communication, a two-step strategy, first including pure culture production and the second including ready-to-use culture production of the biocontrol agent is disclosed. The production costs and success of this technology in RSW management of oil palm are also discussed.

'Candidatus Liberibacter brunswickensis' colonization has no effect to the early development of Solanum melongena.

This study is the first to investigate the presence and movement of the novel Liberibacter species 'Candidatus Liberibacter brunswickensis' (CLbr) in eggplant, Solanum melongena. The psyllid, Acizzia solanicola can transmit CLbr to eggplant and CLbr can be acquired by CLbr-negative A. solanicola individuals from CLbr-positive eggplants. In planta, CLbr can replicate, move and persist. Investigation into the early development of eggplants showed that CLbr titres had increased at the inoculation site at 14 days post inoculation access period (DPIAP). CLbr had become systemic in the majority of plants tested by 28 DPIAP. The highest bacterial titres were recorded at 35 DPIAP in all samples of the inoculated leaf, the roots, stems and the midrib and petiole samples of the newest leaf (the top leaf). This finding strongly suggests that CLbr movement in planta follows the source to sink relationship as previously described for 'Ca. Liberibacter asiaticus' (CLas) and 'Ca. Liberibacter solanacearum' (CLso). No symptoms consistent with Liberibacter-associated diseases were noted for plants colonised by CLbr during this study, consistent with the hypothesis that CLbr does not cause disease of eggplant during the early stages of host colonisation. In addition, no significant differences in biomass were found between eggplant colonised with CLbr, compared to those that were exposed to CLbr-negative A. solanicola, and to control plants.

Biocontrol potential of plant growth-promoting rhizobacteria against plant disease and insect pest.

Biological control is a promising approach to enhance pathogen and pest control to ensure high productivity in cash crop production. Therefore, PGPR biofertilizers are very suitable for application in the cultivation of tea plants (Camellia sinensis) and tobacco, but it is rarely reported so far. In this study, production of a consortium of three strains of PGPR were applied to tobacco and tea plants. The results demonstrated that plants treated with PGPR exhibited enhanced resistance against the bacterial pathogen Pseudomonas syringae (PstDC3000). The significant effect in improving the plant's ability to resist pathogen invasion was verified through measurements of oxygen activity, bacterial colony counts, and expression levels of resistance-related genes (NPR1, PR1, JAZ1, POD etc.). Moreover, the application of PGPR in the tea plantation showed significantly reduced population occurrences of tea green leafhoppers (Empoasca onukii Matsuda), tea thrips (Thysanoptera:Thripidae), Aleurocanthus spiniferus (Quaintanca) and alleviated anthracnose disease in tea seedlings. Therefore, PGPR biofertilizers may serve as a viable biological control method to improve tobacco and tea plant yield and quality. Our findings revealed part of the mechanism by which PGPR helped improve plant biostresses resistance, enabling better application in agricultural production.

Comparative genomics of the carmine cochineal symbiont Candidatus Dactylopiibacterium carminicum reveals possible protection to the host against viruses via CRISPR/Cas.

We present new genomes from the bacterial symbiont Candidatus Dactylopiibacterium carminicum obtained from non-domesticated carmine cochineals belonging to the scale insect Dactylopius (Hemiptera: Coccoidea: Dactylopiidae). As Dactylopiibacterium has not yet been cultured in the laboratory, metagenomes and metatranscriptomics have been key in revealing putative symbiont functions. Dactylopiibacterium is a nitrogen-fixing beta-proteobacterium that may be vertically transmitted and shows differential gene expression inside the cochineal depending on the tissue colonized. Here we found that all cochineal species tested had Dactylopiibacterium carminicum which has a highly conserved genome. All Dactylopiibacterium genomes analyzed had genes involved in nitrogen fixation and plant polymer degradation. Dactylopiibacterium genomes resemble those from free-living plant bacteria, some found as endophytes. Notably, we found here a new putative novel function where the bacteria may protect the insect from viruses, since all Dactylopiibacterium genomes contain CRISPRs with a spacer matching nucleopolyhedrovirus that affects insects.

Rickettsia induces strong cytoplasmic incompatibility in a predatory insect.

Rickettsia, a group of intracellular bacteria found in eukaryotes, exhibits diverse lifestyles, with some acting as vertebrate pathogens transmitted by arthropod vectors and others serving as maternally transmitted arthropod endosymbionts, some of which manipulate host reproduction for their own benefit. Two phenotypes, namely male-killing and parthenogenesis induction are known as Rickettsia-induced host reproductive manipulations, but it remains unknown whether Rickettsia can induce other types of host manipulation. In this study, we discovered that Rickettsia induced strong cytoplasmic incompatibility (CI), in which uninfected females produce no offspring when mated with infected males, in the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular phylogenetic analysis revealed that the Rickettsia strain was related to Rickettsia bellii, a common insect endosymbiont. Notably, this strain carried plasmid-encoded homologues of the CI-inducing factors (namely cifA-like and cifB-like genes), typically found in Wolbachia, which are well-known CI-inducing endosymbionts. Protein domain prediction revealed that the cifB-like gene encodes PD-(D/E)XK nuclease and deubiquitinase domains, which are responsible for Wolbachia-induced CI, as well as ovarian tumour-like (OTU-like) cysteine protease and ankyrin repeat domains. These findings suggest that Rickettsia and Wolbachia endosymbionts share underlying mechanisms of CI and that CI-inducing ability was acquired by microbes through horizontal plasmid transfer.

Differential expression of "Candidatus Liberibacter solanacearum" genes and prophage loci in different life stages of potato psyllid.

Psyllid species, including the potato psyllid (PoP) Bactericera cockerelli (Sulc) (Triozidae) serve as host and vector of "Candidatus Liberibacter spp." ("Ca. Liberibacter"), which also infects diverse plant hosts, including citrus and tomato. Psyllid transmission of "Ca. Liberibacter" is circulative and propagative. The time of "Ca. Liberibacter" acquisition and therefore vector life stage most competent for bacterial transmission varies by pathosystems. Here, the potato psyllid-"Ca. Liberibacter solanacearum" (CLso) pathosystem was investigated to dissect CLso-prophage interactions in the tomato plant and PoP-psyllid host by real-time quantitative reverse transcriptase amplification of CLso genes/loci with predicted involvement in host infection and psyllid-CLso transmission. Genes/loci analyzed were associated with (1) CLso-adhesion, -invasion, -pathogenicity, and -motility, (2) prophage-adhesion and pathogenicity, and (3) CLso-lysogenic cycle. Relative gene expression was quantified by qRT-PCR amplification from total RNA isolated from CLso-infected 1st-2nd and 4th-5th nymphs and teneral adults and CLso-infected tomato plants in which CLso infection is thought to occur without SC1-SC2 replication. Gene/loci expression was host-dependent and varied with the psyllid developmental stage. Loci previously associated with repressor-anti-repressor regulation in the "Ca Liberibacter asiaticus"-prophage pathosystem, which maintains the lysogenic cycle in Asian citrus psyllid Diaphorina citri, were expressed in CLso-infected psyllids but not in CLso-infected tomato plants.

Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus (CLas),' the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Improved control of Trialeurodes vaporariorum using mixture combinations of entomopathogenic fungi and the chemical insecticide spiromesifen.

Greenhouse whitefly (Trialeurodes vaporariorum) is a major global pest, causing direct damage to plants and transmitting viral plant diseases. Management of T. vaporariorum is problematic because of widespread pesticide resistance, and many greenhouse growers rely on biological control agents to regulate T. vaporariorum populations. However, these are often slow and vary in efficacy, leading to subsequent application of chemical insecticides when pest populations exceed threshold levels. Combining chemical and biological pesticides has great potential but can result in different outcomes, from positive to negative interactions. In this study, we evaluated co-applications of the entomopathogenic fungi (EPF) Beauveria bassiana and Cordyceps farinosa and the chemical insecticide spiromesifen in laboratory bioassays. Complex interactions between the EPFs and insecticide were described using an ecotoxicological mixtures model, the MixTox analysis. Depending on the EPF and chemical concentrations applied, mixtures resulted in additivity, synergism, or antagonism in terms of total whitefly mortality. Combinations of B. bassiana and spiromesifen, compared to single treatments, increased the rate of kill by 5 days. Results indicate the potential for combined applications of EPF and spiromesifen as an effective integrated pest management strategy and demonstrate the applicability of the MixTox model to describe complex mixture interactions.

A Transmission Assay of 'Candidatus Liberibacter asiaticus' Using Citrus Phloem Sap and Topical Feeding to Its Insect Vector, Diaphorina citri.

'Candidatus Liberibacter asiaticus', the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, 'Ca. L. asiaticus' is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of 'Ca. L. asiaticus' becomes available.

Facultatively intrabacterial localization of a planthopper endosymbiont as an adaptation to its vertical transmission.

Transovarial transmission is the most reliable way of passing on essential nutrient-providing endosymbionts from mothers to offspring. However, not all endosymbiotic microbes follow the complex path through the female host tissues to oocytes on their own. Here, we demonstrate an unusual transmission strategy adopted by one of the endosymbionts of the planthopper Trypetimorpha occidentalis (Hemiptera: Tropiduchidae) from Bulgaria. In this species, an Acetobacteraceae endosymbiont is transmitted transovarially within deep invaginations of cellular membranes of an ancient endosymbiont Sulcia-strikingly resembling recently described plant virus transmission. However, in males, Acetobacteraceae colonizes the same bacteriocytes as Sulcia but remains unenveloped. Then, the unusual endobacterial localization of Acetobacteraceae observed in females appears to be a unique adaptation to maternal transmission. Further, the symbiont's genomic features, including encoding essential amino acid biosynthetic pathways and its similarity to a recently described psyllid symbiont, suggest a unique combination of the ability to horizontally transmit among species and confer nutritional benefits. The close association with Acetobacteraceae symbiont correlates with the so-far-unreported level of genomic erosion of ancient nutritional symbionts of this planthopper. In Sulcia, this is reflected in substantial changes in genomic organization, reported for the first time in the symbiont renowned for its genomic stability. In Vidania, substantial gene loss resulted in one of the smallest genomes known, at 108.6 kb. Thus, the symbionts of T. occidentalis display a combination of unusual adaptations and genomic features that expand our understanding of how insect-microbe symbioses may transmit and evolve.IMPORTANCEReliable transmission across host generations is a major challenge for bacteria that associate with insects, and independently established symbionts have addressed this challenge in different ways. The facultatively endobacterial localization of Acetobacteraceae symbiont, enveloped by cells of ancient nutritional endosymbiont Sulcia in females but not males of the planthopper Trypetimorpha occidentalis, appears to be a unique adaptation to maternal transmission. Acetobacteraceae's genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates the apparent consequences of such close association. Combined, this multi-partite symbiosis expands our understanding of the diversity of strategies that insect symbioses form and some of their evolutionary consequences.

Distribution and dynamic changes of Huanglongbing pathogen in its insect vector Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama is the leading vector of Candidatus Liberibacter asiaticus (CLas), the causative agent of citrus Huanglongbing (HLB) disease. The distribution and dynamics of CLas within ACP are critical to understanding how the transmission, spread and infection of CLas occurs within its host vector in nature. In this study, the distribution and titer changes of CLas in various tissues of ACP 5th instar nymphs and adults were examined by fluorescence in situ hybridization (FISH) and real-time quantitative PCR (qPCR) techniques. Results demonstrated that 100% of ACP 5th instar nymphs and adults were infected with CLas following feeding on infected plants, and that CLas had widespread distribution in most of the tissues of ACP. The titers of CLas within the midgut, salivary glands and hemolymph tissues were the highest in both 5th instar nymphs and adults. When compared with adults, the titers of CLas in these three tissues of 5th instar nymphs were significantly higher, while in the mycetome, ovary and testes they were significantly lower than those of adults. FISH visualization further confirmed these findings. Dynamic analysis of CLas demonstrated that it was present across all the developmental ages of ACP adults. There was a discernible upward trend in the presence of CLas with advancing age in most tissues of ACP adults, including the midgut, hemolymph, salivary glands, foot, head, cuticula and muscle. Our findings have significant implications for the comprehensive understanding of the transmission, dissemination and infestation of CLas, which is of much importance for developing novel strategies to halt the spread of CLas, and therefore contribute to the efficient prevention and control of HLB.

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.