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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Rickettsia symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating.

Heritable symbionts are common among animals in nature, but the molecular mechanisms underpinning symbiont invasions of host populations have been elusive. In this study, we demonstrate the spread of Rickettsia in an invasive agricultural pest, the whitefly Bemisia tabaci Mediterranean (MED), across northeastern China from 2018 to 2023. Here, we show that the beneficial symbiont Rickettsia spreads by manipulating host hormone signals. Our analyses suggest that Rickettsia have been horizontally acquired by B. tabaci MED from another invasive whitefly B. tabaci Middle East-Asia Minor 1 during periods of coexistence. Rickettsia is transmitted maternally and horizontally from female B. tabaci MED individuals. Rickettsia infection enhances fecundity and results in female bias among whiteflies. Our findings reveal that Rickettsia infection stimulates juvenile hormone (JH) synthesis, in turn enhancing fecundity, copulation events, and the female ratio of the offspring. Consequently, Rickettsia infection results in increased whitefly fecundity and female bias by modulating the JH pathway. More female progeny facilitates the transmission of Rickettsia. This study illustrates that the spread of Rickettsia among invasive whiteflies in northeastern China is propelled by host hormone regulation. Such symbiont invasions lead to rapid physiological and molecular evolution in the host, influencing the biology and ecology of an invasive species.

Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

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.

A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes the in vitro gene expression with bacterial ribosomes.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2-20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.