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      Ultrastructural Exploration on the Histopathological Change in Phenacoccus fraxinus Infected with Lecanicillium lecanii

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          Abstract

          The histopathological changes of the second instar nymph of the mealybug Phenacoccus fraxinus infected with Lecanicillium lecanii strain 3.4505 were investigated using light, scanning and transmission electron microscopy. The results demonstrated that L. lecanii 3.4505 could infect P. fraxinus in a short period. At 24 h post-inoculation, the conidia of L. lecanii 3.4505 adhered to the indented gloves or intersegmental folds of the insect body surface. Subsequently, the germinated conidia produced germ-tubes, appressoria and extended hyphae, which tightly adhered to the cuticle. Penetration of cuticle could be achieved either by peg form appressoria or directly by hyphae. Also, the conidia and hyphae could secrete massive mucilages causing visible damage to the host cuticle. After 48 h, the body wall, tissues and organs, including cuticle, trachea, fat body, muscle, Malpighian tubules and nerve ganglion, were destroyed by ramification of hyphae as a result of infection. The endoplasmic reticulum hypertrophied and formed obvious fingerprint agglomerates, and the mitochondria swelled and deformed in the haemocytes. Finally, the mycelium fully occupied the entire haemocoel. The entire bodies were wrapped in a white mycelium, with the mycelium extending radically outward.

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          Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during Fungal Invasion of Host (Manduca sexta) Cuticle.

          Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.
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            [Effect of multi-generation culture on virulence of Lecanicilliurn lecanii in different media].

            The strain No. V3.4504 of Lecanicilliurn lecanii (Zimmermann), an entomopathogenic fungus, was studied on the effect of successive multi-generation culture in seven different media on its colony growth characteristics, extracellular enzyme activities and the virulence against scale insects. The strain No. V3.4504 of L. lecanii was original isolated from a natural infected scale insect. The two species of scale insects used were Rhodococcus sariuoni Borchsenius and Ceroplastes japonicus Green. Seven media were used and fungus colony characteristics, growth rate and sporulation, extracellular protease and chitinase activity, and infective effect against the two species of scale insects were conducted. The fungus cultured on PDA medium for successive nine generations showed the most fast in colony growth, the minimal in sporulation, straight decline of extracellular protease and chitinase activity with generation increasing, and the minimal mortality of the scale insects. There was no significant effect to promote virulence of the fungus by increasing peptone into medium. On the media D, E and F, that with the body materials of the two scale insects, although the fungus appeared lower in the colony growth rate, its sporulation was higher upward 8.83 x 10(6) - 9.13 x 10(6) spores/cm2, extracellular protease and chitinase activities averagely reached 2.16 - 2.13 U/g and 1.01 - 1.03 U/g respectively, and the mortalities of the two scale insects were 55% - 58% and 39% - 42% respectively. Cultured three generations in vitro of the two scale insects, the fungus exhibited the highest activities in its protease and chitinase that were 3.08 - 2.92 U/g and 1.45 - 1.42 U/g respectively and the best infection effect against the two scale insects with mortalities of 71.30% and 58.89% respectively. A linear correlation was found between extracellular protease and chitinase activities of the fungus and the mortalities of the scale insects. Cultured on PDA medium successive multiple generations made retrogradation of the strain No. V3.4504 of of L. lecanii. It was significant effect on keeping the vigor and higher virulence of the fungus adding the body materials of the scale insects into the medium. The vitro by using live scale insects as medium materials was the best way for the rejuvenation of the entomopathogenic fungus and promoting its virulence.
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              [Reliability of extracellular protease and lipase activities of Beauveria bassiana isolates used as their virulence indices].

              M. Feng (1998)
              The extracellular protease and lipase activities of 17 Beauveria bassiana isolates from different hosts and countries were evaluated for the reliability for the indices of their virulence to the migratory grasshopper, Melanoplus sanguinipes. Virulence assay of each isolate included about 30 10-d-old grasshoppers receiving topical inoculation with the suspension of 10(7) conidia/ml. In the assays of the enzymes, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and p-nitrophenyl palmitate were used as a substrate to measure the activities of protease (3 replicates) and lipase (4 replicates) in the filtrates of gelatin-based and sunflower oil-based liquid cultures of each isolate, respectively. Varying among the isolates assayed, the estimates of LT50's, protease units (PU), and lipase units (LU) were 5.27-16.89 d, 0.47-3.37 x 10(-2) mumol.ml-1.min-1, and 0.00-56.75 mumol.ml-1.h-1, respectively. Regression analysis revealed that PU was significantly (P 0.10). Based on the determination coefficients (r2) from the regression, PU alone interpreted at most 67% of the variation in the mortality 7d after inoculation but less than 50% in most of the days considered and only 38% in LT50's. Thus, the author suggested that PU could be used as virulence index only for early-stage selection of candidate isolates in large quantity and could not entirely replace conventional virulence assay method that remains most reliable.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                28 January 2015
                2015
                : 10
                : 1
                : e0117428
                Affiliations
                [1 ]School of Life Science, Shanxi University, Taiyuan, China
                [2 ]Institute of Applied Biology, Shanxi University, Taiyuan, China
                UMR INRA/INSA, BF2I, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YG YX JX. Performed the experiments: YG. Analyzed the data: YG WL. Contributed reagents/materials/analysis tools: YX QX WL. Wrote the paper: YG YX.

                Article
                PONE-D-14-13914
                10.1371/journal.pone.0117428
                4309582
                25629309
                6f64fcee-ffeb-4fd0-a0c8-925f4d1f0c66
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 18 April 2014
                : 23 December 2014
                Page count
                Figures: 5, Tables: 0, Pages: 9
                Funding
                This research was supported by the National Natural Science Foundation of China (30671693 and 31070584), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20101401110008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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