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Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

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Drug-resistant bacteria constitute a big barrier against current pharmacotherapy. Efforts are urgent to discover antibacterial drugs with novel chemical and biological features. Our work aimed at the synthesis, evaluation of antibacterial effects, and toxicity of licochalcone C (LCC), a naturally occurring chalcone. The synthetic route included six steps, affording a 10% overall yield. LCC showed effects against Gram-positive bacteria (MIC = 6.2-50.0 µg/mL), Mycobacterium species (MIC = 36.2-125 µg/mL), and Helicobacter pylori (MIC = 25 µg/mL). LCC inhibited the biofilm formation of MSSA and MRSA, demonstrating MBIC50 values of 6.25 µg/mL for both strains. The investigations by fluorescence microscopy, using PI and SYTO9 as fluorophores, indicated that LCC was able to disrupt the S. aureus membrane, similarly to nisin. Systemic toxicity assays using Galleria mellonella larvae showed that LCC was not lethal at 100 µg/mL after 80 h treatment. These data suggest new uses for LCC as a compound with potential applications in antibacterial drug discovery and medical device coating.

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Leaf-cutting ants are the most important pests in several cropping systems in the Neotropics. Granulated baits containing active ingredients, considered hazardous by the Stockholm Convention, are the usual method to control these ants. Isocycloseram is a new insecticide molecule with high safety margin for mammals, but without registration for the ants in general. Thus, this study investigated the effectiveness of granulated baits with isocycloseram in leaf-cutting ants control under laboratory and field conditions. Initially, the mortality of Atta sexdens workers, fed with dehydrated citrus pulp paste containing different concentrations of isocycloseram was evaluated in the laboratory for 21 days, for toxicological classification. Subsequently, the loading, devolution, and incorporation of baits with different concentrations of isocycloseram and the mortality of A. sexdens colonies were evaluated in the laboratory. After that, the percentages of loading and devolution of baits, foraging activity, and colony mortality treated with 0.05, 0.1, 0.2, and 0.3% of isocycloseram were evaluated for the species A. sexdens, A. laevigata, and Acromyrmex lundii in field conditions. All concentrations of isocycloseram killed more than 15% of ants in 24 h and more than 90% in 21 days in the laboratory, being classified as a fast-acting and highly effective active ingredient. Baits with 0.001 to 0.03% of isocycloseram were highly loaded and exhibited low rate of devolution. The mortality of A. sexdens colony was higher at concentrations between 0.075 and 0.3%, in the laboratory. Baits containing isocycloseram at concentrations of 0.2 and 0.3% were highly loaded, presented low devolution rates, and were highly efficient in controlling A. sexdens, A. laevigata, and A. lundii in the field, at dosages of 6, 10, and 12 g/m² of nest. This is the first report of the use of isocycloseram against leaf-cutting ants, contributing to the development of efficient and toxicologically safer ant baits.

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AIMS: Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. METHODS AND RESULTS: In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml-1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. CONCLUSION: The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri, and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri.

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Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae) is a colonizer of cadavers in the Neotropical Region. Nevertheless, data on development for the P. (P.) chrysostoma (e.g., instar duration) and behavioral strategies used by the species for locating and colonizing a corpse are scant. We aimed to explore bionomic and reproductive aspects of the flesh fly P. (P.) chrysostoma, and in this article we: (a) provide quantitative data on the life cycle of P. (P.) chrysostoma; (b) present bionomic measurements (length and weight) of larvae and pupae; (c) describe intrauterine egg and larvae development; and (d) analyze the ovo/larviposition behavior by gravid females. Females showed ovaries with discernible eggs and larvae between 8 and 10 days (x̅ = 23.3 eggs/female). This study reports the first observation of egg deposition, an atypical behavior for the species. The average development time for immature stages was 22.24 h and 21.36 h for 1st and 2nd respectively, and 3rd showed an average development time of 80.47 h. Pupa had the longest duration (x̅ = 295.69 h). A direct increase was observed in weight (P < 0.05) and length (P < 0.05) throughout time. The average survival time of males and females is approximately 30 days. This study expands the knowledge on P. (P.) chrysostoma, such as facultative ovoviviparity under laboratory conditions and the life cycle, which may benefit future studies for accuracy in entomology-based estimation of minimum post-mortem interval (min PMI).

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Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of X. citri. It was observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC exhibited a delayed onset of citrus canker symptoms compared with the wild-type X. citri. Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in X. citri virulence.

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This study aimed to investigate the phytochemistry of lemongrass (Cymbopogon citratus) inoculated with Azospirillum brasilense and grown in lead (Pb)-contaminated soil to assess its responses to inoculation under different Pb levels. The experimental design was completely randomized in a 2 × 5 factorial scheme: two levels of A. brasilense (absence or presence) and five Pb levels. After four months of treatment, the following were analyzed: total and reducing sugars, total phenolic content, flavonoids, antioxidant activity, antioxidant enzymes, proline, and essential oil (EO) content and composition. Soil Pb levels and A. brasilense inoculation affected phytochemicals in lemongrass plants. Azospirillum inoculation reduced total sugars in the roots at all soil Pb levels, while increasing Pb levels favored a rise in sugar contents. There was an increase in flavonoid content in treatments associated with Pb and inoculated with A. brasilense. Antioxidant capacity was lower at lower Pb levels, regardless of bacterial inoculation. Enzymatic response was mainly affected by Pb concentrations between 50 and 100 mg kg-1 soil. EO content was influenced by soil Pb levels, with higher EO production at 500 mg Pb kg-1 soil and without A. brasilense inoculation. Overall, lemongrass cultivation in Pb-contaminated areas can be an alternative to phytoremediation and EO production for the industry.

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Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312 mg L-1, followed by 625 mg L-1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15 min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection.

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Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

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Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 µg mL-1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 µg mL-1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. KEY POINTS: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease.

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