Detection of Aflatoxin B1 in Silkworm Larvae Attacked by an Aspergillus flavus Isolate from a Sericultural Farm

Aflatoxin B₁ (0.05 μM per larvae) was detected in silkworm larvae artificially attacked by an Aspergillus flavus isolate from a sericultural farm in Japan.     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFR-TKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.     

Expression of Xylella fastidiosa Fimbrial and Afimbrial Proteins during Biofilm Formation

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.Aggregative growth is a common feature in the microbial world, and its discovery radically changed our concept of microbial growth dynamics. A cellular aggregate adhering to a surface is known as a biofilm. It has important characteristics, such as greater resistance to antimicrobial compounds (34, 54), increased capacity of the cells to take up nutrients from the environment (59), and higher detoxification efficiency resulting from an increase in expression of genes encoding efflux pumps (43). These characteristics give the biofilm cells a great adaptive advantage.Biofilm growth also confers advantages to plant pathogens by promoting virulence and protection against plant defense responses. Bacteria can colonize different niches in the plant, from aerial surfaces to roots and the vascular system, and biofilm formation can play a role at all of these sites of colonization. In the vessels, biofilms are very important since the cells need to survive in a competitive habitat where plant defense compounds are produced in response to infection (7).Biofilm development is divided into at least the following five phases: (i) reversible attachment, (ii) irreversible attachment, (iii) beginning of maturation, (iv) mature biofilm, and (v) dispersion (13, 50). In Xylella fastidiosa strain 9a5c, the maturation phase occurs between days 15 and 20 in vitro, while dispersion occurs between days 25 and 30, as observed by our analysis of biofilm formation using different methods, including scanning electron microscopy and quantification of exopolysaccharides, biomass, and the total protein (unpublished data). The establishment and development of biofilms of plant-colonizing bacteria share several features with the establishment and development of biofilms of human bacterial pathogens, such as regulation by quorum sensing, nutrient starvation regulation, and phase variation. Motility is also an important factor not only for the initiation and development of the biofilm but also for dispersion (50). Attachment is mediated by surface-associated structures, which include both polysaccharides and proteins classified as fimbrial and afimbrial adhesins, depending on the structure to which they contribute. Fimbrial adhesins form filamentous structures, while afimbrial adhesins produce projections on the outer membrane (23).X. fastidiosa, a Gram-negative phytopathogen that grows as a biofilms in both plant xylem vessels and the cybarium of insect vectors, is a major threat to plant production around the world. In Brazil, it has a major economic impact on citriculture since it causes citrus variegated chlorosis disease (CVC) (35, 39, 42). The biofilm formed by X. fastidiosa blocks the xylem vessels of susceptible citrus plants, impairing water flow. This blockage leads to a drastic reduction in fruit size (32) and, consequently, severe economic losses resulting from reduced plant productivity (4).Due to the economic damage caused by CVC, there has been a major effort to generate more information about its biology. This led to sequencing of the genome of the pathogen. The X. fastidiosa genome harbors a wide variety of genes encoding adhesins (53). Bacterial cell surface adhesins are important in the initial phases of adherence to surfaces, as well as in bacterium-bacterium interactions and microcolony development (15). Insight into X. fastidiosa has also come from genome analysis of a strain of X. fastidiosa which causes Pierce''s disease of grape (58). Studies of this strain showed that the cellular aggregation process involves type I and type IV fimbrial adhesins. The two types of fimbriae present different adhesion forces that help bacteria adhere to a substrate (10, 30). Adhesion proteins have also been demonstrated to mediate adherence to carbohydrates of leafhopper foregut surfaces (27). In addition, both fimbrial and afimbrial adhesins are important for plant pathogenicity (38, 41). However, the expression of these proteins during X. fastidiosa biofilm formation either in vitro or in planta is still poorly understood. For X. fastidiosa strains causing CVC, nothing is known about the role of these proteins in pathogenicity or biofilm formation, although some adhesion-encoding genes, such as pilA2, pilC, xadA1, and xadA2, were found to be upregulated either in virulent strains of X. fastidiosa or during biofilm formation (12, 14). These results suggest that the biofilm mode of growth is important for successful colonization of the citrus host by X. fastidiosa strains that cause CVC. In this work we focused on the temporal expression of the PilA2 and PilC fimbrial proteins and XadA1 and XadA2, which are afimbrial adhesins, during in vitro development of X. fastidiosa CVC biofilms. We demonstrated that the temporal and spatial patterns of expression of the fimbrial and afimbrial adhesins are very different during biofilm development in vitro. Moreover, we also verified that these adhesins are present in X. fastidiosa cells in symptomatic plants of three different hosts (citrus, periwinkle, and hibiscus).     

Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants

AIMS: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome. METHODS AND RESULTS: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. CONCLUSIONS: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.     

N-Acetylcysteine in Agriculture,a Novel Use for an Old Molecule: Focus on Controlling the Plant–Pathogen Xylella fastidiosa

Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria.     

Morphology of lymphatics of the mammalian heart with special reference to the architecture and distribution of the subepicardial lymphatic system

The subepicardial lymphatic system in the rat and dog heart has been investigated by means of scanning electron microscopy. Following application of hydrogen peroxide, the epicardium was removed with a forceps under a dissecting microscope. The subepicardial region contained a well-developed lymphatic system which consisted of the main lymphatic trunks and lymphatic capillaries. The lymphatic trunks of large diameters ran from the apex of the heart to its base. The subepicardial lymphatic capillaries were ramified and anastomosed with each other to form a relatively dense network which extended over the entire surface of both ventricles. These networks joined the main lymphatic trunks. Further, some similar networks were connected with the underlying myocardial lymphatic capillaries.