Expression of pathogenesis-related genes in Metarhizium anisopliae when infecting Spodoptera exigua

Characterization of pathogenesis genes of Metarhizium anisopliae, will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24 h after infection, when the infected larvae reached the moribund stage (36 h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2 h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12 h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1, CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1, CHI2, and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect.     

Evaluation of Metarhizium anisopliae strains as potential biocontrol agents of the tick Rhipicephalus (Boophilus) microplus and the cotton stainer Dysdercus peruvianus

The negative aspects of traditional pest control have led to the investigation of alternative methods such as biological control. Metarhizium anisopliae, well known as an entomopathogenic fungus capable of actively invading and killing its hosts and thus a candidate biopesticide, is here tested against two agricultural pests of economic/social importance and also evaluated for its chitinolytic secretion and capacity to grow and sporulate at different temperatures. None of the isolates was able to grow below 4 °C or above 37 °C. Chitinolytic activity under artificial growth conditions revealed that Rhipicephalus (Boophilus) microplus cuticle induces N-acetyl-β-d-glucosaminidase and endochitinase activities more efficiently than Dysdercus peruvianus cuticle and that glucose did not repress those activities. Bioassays were carried out with R. microplus females and fourth instar D. peruvianus. Six isolates of M. anisopliae were pathogenic to the engorged female cattle ticks. E6, GC47 and CG97 were the most virulent isolates for both arthropod models although differences were seen among them. M. anisopliae strains caused 90–100 % mortality on the fourth post-infection day in R. microplus. D. peruvianus females were more sensitive to fungal infection than males, and the most virulent strains caused 50 % mortality on the third to fourth day post-infection. Our studies suggest that M. anisopliae strain CG47 is a candidate for commercial pesticide formulations due to its capacity to kill both hosts and its ability to sporulate at higher temperatures.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Colonisation dynamics and virulence of two clonal groups of multidrug-resistant Escherichia coli isolated from dogs

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) β-lactamase. CG1 isolates (n = 14) possessed two of 36 assayed extraintestinal virulence genes (iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates (n = 19) contained four such genes (iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40 days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.     

Influence of ligands on the fac⇌Δhνmer isomerization in [RuCl3(NO)(P–P)] complexes, [P–P = R2P(CH2)nPR2 (n = 1–3) and R2P(CH2)POR2, PR2–CHCH–PR2, R = Ph and (C6H11)2P-(CH2)2-P(C6H11)2]

[RuCl₃(NO)(P–P)], [P–P = R₂P(CH₂)_nPR₂ (n = 1–3) and R₂P(CH₂)POR₂, PR₂–CHCH–PR₂, R = Ph and (C₆H₁₁)₂P-(CH₂)₂-P(C₆H₁₁)₂] were obtained and characterized by ³¹P {¹H} NMR, IR spectroscopies and cyclic voltammetry. The structures of fac-[RuCl₃(NO)(P–P)], P–P = dppm (1), dppe (2), c-dppen (3) and dppp (4), mer-[RuCl₃(NO)(dcpe)] (6a) and mer-[RuCl₃(NO)(dppmO)] (7) have been determined by X-ray diffraction. Photochemical isomerization of fac- to mer-[RuCl₃(NO)(P–P)] was observed under white light in a CH₂Cl₂ solution and in solid state. The isomerization processes were followed by IR and ³¹P {¹H} spectra. The mer-[RuCl₃(¹⁵NO)(dppb)] isomer was used for the definition of the phosphorus atoms in the structure of the complex in solution. The electrochemical study shows that the oxidation/reduction processes observed in these complexes are dependent on both the isomer (fac or mer) and the solvent. In CH₂Cl₂, the NO⁺ reduction potentials are less negative for the mer-isomers than for the fac ones, while in CH₃CN solvent these potentials are, in general, very close for both isomers.     

A laccase exclusively expressed by Metarhizium anisopliae during isotropic growth is involved in pigmentation,tolerance to abiotic stresses and virulence

Insect pathogenic fungi including Metarhizium anisopliae offer an environmentally friendly alternative to chemical pesticides. However, their use has been limited by their relatively slow killing speed compared to chemicals and low tolerance to abiotic stresses. We report here on a class 1 laccase (MLAC1) that is involved in both virulence and tolerance to environmental stresses. Mlac1 is expressed during isotropic growth (swelling) but not during polarized growth (e.g., germ tubes and hyphae); Mlac1 is therefore expressed exclusively in the later stages of conidiation and in blastospores when M. anisopliae is living as a saprophyte. During infection processes, Mlac1 is also expressed by appressoria (infection structures) on the cuticle surface and hyphal bodies inside the insect haemocoel. Disrupting Mlac1 reduced virulence to caterpillars because of impaired appressoria and delayed post-infection events. It also produced a yellow-conidia phenotype with increased conidial susceptibility to heat shock (45 °C for 2 h) and UV-B stress. The relationship between M. anisopliae’s pigment-synthesis pathway and its adaptation to diverse natural habitats is discussed.     

Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles

Termites adjust their response to entomopathogenic fungi according to the profile of fungal volatile organic compounds (VOCs). This study demonstrates the pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea (=Paecilomyces fumosoroseus) towards the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Using no-choice assays, M. anisopliae was found to be highly virulent (LT₅₀ 3.10 d) when compared to B. bassiana (LT₅₀ 6.62 d) and I. fumosorosea (LT₅₀ 12.39 d). Also using choice assays, the foraging behavior of C. formosanus was determined in the presence of pathogenic fungi. The highly pathogenic fungi (M. anisopliae) elicited a repellent response, causing most of the termites to forage in a safe zone farthest from the fungal source. This repellency resulted in relatively low mortality similar to the controls. The repellency of M. anisopliae conidia can be used to protect human belongings and timber from termites. While I. fumosorosea cultures were not repellent to C. formosanus workers, the termites were highly susceptible to infection. Electroantennographic responses of workers showed approximately 47% and 78% lower level of response to conidia of B. bassiana and I. fumosorosea, respectively, as compared to M. anisopliae. The VOC profile of repellent cultures of M. anisopliae mainly consisted of paraffins (60.97%), while the major proportion of the I. fumosorosea profile consisted of branched and cyclic alkanes (84.41%). From the above findings, we conclude that the incorporation of I. fumosorosea may increase the control potential of bait.     

Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

Intraspecific variability of Pythium myriotylum isolated from cocoyam and other host crops

Intraspecific variability within 51 isolates of Pythium myriotylum from cocoyam (Xanthosoma sagittifolium) and other host crops was analysed using optimum growth temperature, esterase banding patterns, AFLPs, rDNA–ITS sequencing, and virulence to cocoyam. P. myriotylum isolates virulent to cocoyam could easily be differentiated from other isolates of P. myriotylum by their optimum growth temperature. Isolates from cocoyam grew best at 28 °C with no growth at 37 °C, while P. myriotylum isolates from other host crops had their optimum growth temperature at 37 °C. Esterases produced consistent zymograms with 18 discrete esterase markers, but no monomorphic markers were produced for isolates virulent to cocoyam. Isozyme profiles based on esterase analysis showed that isolates that infect cocoyam plantlets formed a related group, irrespective of their geographic origin. P. myriotylum isolates from other host plants also grouped together, but could clearly be distinguished from the cocoyam cluster. AFLPs produced 189 scorable bands for the cocoyam isolates, of which 77 % are monomorphic. Phenetic analysis of AFLP data grouped all isolates originating from cocoyam together except for the isolates C103-04, CMR17, CMR22, and CMR25. These isolates regrouped with isolates of Pythium myriotylum from other host crops or the outgroup and were found not to be pathogenic for cocoyam. ITS sequences of isolates of P. myriotylum from cocoyam were 99.1–99.7 % identical to sequences deposited in GenBank. However, alignments of ITS sequences revealed a base transition at position 824 from adenine in typical isolates of P. myriotylum to guanine in isolates that could infect cocoyam plantlets. In a limited pathogenicity test, all isolates from cocoyam having guanine at position 824 were able to infect tissue culture derived cocoyam but not those exhibiting adenine. This study demonstrates for the first time, molecular evidence that isolates of P. myriotylum that infect cocoyam are distinct from P. myriotylum isolates from other crops and have developed a certain degree of host adaptation.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.     

Nigrosphaerin A a new isochromene derivative from the endophytic fungus Nigrospora sphaerica

Nigrosphaerin A, a new isochromene derivative (1), was isolated from the endophytic fungus Nigrospora sphaerica and chemically identified as 3-(3,4-dihydroxyphenyl)-4,6,8-trihydroxy-1H-isochromen-1-one-6-O-β-d-glucopyranoside. In addition nineteen known compounds (2–20) were isolated from the same fungus and chemically identified. Compounds (1–3, 5, and 7–16) were isolated for the first time from this fungus. In vitro antileukemic, antileishmanial, antifungal, antibacterial and antimalarial activities of (1–20) were examined. Compounds 5, 7, 9 and 10 showed good antileukemic activity against HL60 cells with IC₅₀ values of 0.03, 0.39, 0.2 and 0.4 μg/mL, respectively and against K562 cells with IC₅₀ values of 0.35, 0.35, 0.49 and 0.01 μg/mL, respectively. Compounds 3, 4 and 6 showed moderate antileishmanial activity with IC₅₀ values of 30.2, 26.4 and 36.4 μg/ml, respectively. Compound 7 showed moderate antifungal activity against Cryptococcus neoformans with IC₅₀ value of 14.8 μg/mL.     

Can Beauveria bassiana Bals. (Vuill) (Ascomycetes: Hypocreales) and Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) be used together for improved biological control of Bactericera cockerelli (Hemiptera: Triozidae)?

Bactericera cockerelli (Sulc.) is an important pest of solanaceous crops and a vector of the pathogen Candidatus Liberibacter psyllaurous. Biocontrol of this pest has been attempted with either entomopathogenic fungi or the parasitoid Tamarixia triozae (Burks), but prior to this study, their potential impact in combination had not been studied. The aim of the present study was to evaluate T. triozae parasitism rates on B. cockerelli nymphs that were previously infected for different periods of time by three isolates of Beauveria bassiana (Bals.) Vuill. Two native isolates (BB40 and BB42) and one commercial isolate (GHA) were used. The virulence of these isolates was first estimated against B. cockerelli and T. triozae. LC₅₀ values for the native isolates BB40 and BB42 against B. cockerelli were 9.5 × 10⁵ and 2.42 × 10⁶ conidia mL⁻¹ respectively; they were significantly more virulent than isolate GHA with an LC₅₀ of 1.97 × 10⁷ conidia mL⁻¹. However, isolate GHA was significantly more virulent against T. triozae with an LC₅₀ of 1.11 × 10⁷ conidia mL⁻¹ compared with LC₅₀s of 1.49 × 10⁷ and 1.14 × 10⁸ conidia mL⁻¹ for the native isolates BB40 and BB42 respectively. Groups of nymphs were then inoculated with LC₂₀, LC₅₀ or LC₉₀ concentrations of each isolate and presented to T. triozae as hosts either on the day of inoculation or 1, 2, 3, 4, 5, 6 days after inoculation. Subsequent levels of parasitism were recorded. Overall, parasitism rates were similar in inoculated and control nymphs. No parasitism occurred in nymphs 6 days after fungal inoculation. Parasitoids used to parasitize uninoculated B. cockerelli nymphs survived significantly longer (7.8 days) than parasitoids that had been used to parasitize fungus-inoculated nymphs (7.3 days). This suggests an inability of the parasitoid to avoid infection when foraging on inoculated nymphs. In conclusion, although the parasitism rate in control and fungus-treated nymphs was similar, suggesting a combination of both biological control agents is possible, we believe there are also negative implications for the parasitoid because its survival was greatly reduced after attacking infected nymphs.     

Diversity of moxifloxacin resistance during a nosocomial outbreak of a predominantly ribotype ARU 027 Clostridium difficile diarrhea

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB. All the laboratory isolates, including the historical ARU 027 isolate Cd196, were susceptible to moxifloxacin (≤2 μg/mL). 13 field isolates were susceptible to ≤2 μg/mL. Five were resistant to from 4 to 12 μg/mL (moderate resistance); 16 were resistant to ≥16 μg/mL (high resistance). We sequenced the quinolone resistance determining regions of gyrA (position 71-460) and gyrB (position 1059-1448) from two susceptible laboratory strains, all five isolates with moderate resistance and two highly resistant isolates. Two highly resistant isolates (Pitt 40, ribotype ARU 027 and Pitt 33, ribotype ARU 001) had the same C245T (Thr₈₂ΔIle) mutation. No other changes were seen. Amplification with primer pairs specific for the C245T mutant gyrA and for the wild type gene respectively confirmed all 16 highly resistant ARU 027 isolates, as well as the highly resistant isolates from other ribotypes, had the C245T mutation and that the mutation was absent from all other isolates. Among the five isolates with moderate resistance we found combinations of mutations within gyrA (T128A, Val₄₃ΔAsp and G349T, Ala₁₁₇ΔSer) and gyrB (G1276A, Arg₄₂₆ΔAsn). The G1396A (Glu₄₆₆ΔLys) mutation was not associated with increased resistance.     

Pentapeptide boronic acid inhibitors of Mycobacterium tuberculosis MycP1 protease

Mycosin protease-1 (MycP₁) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP₁ variants from Mycobacterium thermoresistible (MycP_1mth), Mycobacterium smegmatis (MycP_1msm) and M. tuberculosis (MycP_1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)₂ (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO₂(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC₅₀ values 121.6 ± 25.3 μM for MycP_1mth, 93.2 ± 37.3 μM for MycP_1msm and 37.9 ± 5.2 μM for MycP_1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP₁ catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs.     

3-Anhydro-6-hydroxy-ophiobolin A,a new sesterterpene inhibiting the growth of methicillin-resistant Staphylococcus aureus and inducing the cell death by apoptosis on K562, from the phytopathogenic fungus Bipolaris oryzae

A new ophiobolin derivative, 3-anhydro-6-hydroxy-ophiobolin A (1), as well as two known ophiobolin derivatives 3-anhydro-ophiobolin A (2) and 3-anhydro-6-epi-ophiobolin A (3) were isolated from the PDB culture of a phytopathogenic fungus Bipolaris oryzae. The structure of 1 was elucidated through 2D NMR and other spectroscopic techniques. Compound 1 exhibited strong antimicrobial activity against Bacille Calmette–Guerin, Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus with MIC value of 12.5 μg/mL, and potent antiproliferative activity against cell lines HepG2 and K562 with IC₅₀ of 6.49 μM and 4.06 μM, respectively. Further studies on the cytotoxicity of compound 1 against K562 cells demonstrated that it induced apoptosis, observed by flow cytometric method. Preliminary structure–activity relationships of these ophiobolins and the mechanism of apoptosis induced by 1 were analyzed.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Structure and magnetic properties of a trinuclear nickel(II) complex with benzenetricarboxylate bridge

Novel trinuclear Ni(II) complex [Ni₃(pmdien)₃(btc)(H₂O)₃](ClO₄)₃ · 4H₂O, 1 where pmdien = N,N,N′,N′,N″-pentamethyldiethylenetriamine, H₃btc = 1,3,5-benzenetricarboxylic (trimesic) acid, has been prepared and structurally characterized. Three nickel atoms are bridged by btc trianion and their coordination sphere is completed by three N atoms of pmdien and O atom of the water molecule. The three nickel(II) magnetic centers are equivalent and their coordination spheres are completed to deformed octahedrons. Magnetic susceptibility was measured over the temperature range 1.8–300 K and zJ′ = ?0.19 cm^?1, D = 3.79 cm^?1, g = 2.18 parameters were calculated.     

Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs,Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions

Four entomopathogenic nematode (EPN) species (Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev and Steinernema riobrave Cabanillas, Poinar & Raulston) were tested for virulence against 3^rd instar southern masked chafer white grubs, Cyclocephala lurida Bland. H. bacteriophora and H. megidis, being the most virulent, were selected to evaluate the interaction with an entomopathogenic fungus (EPF), Beauveria bassiana (Balsamo) Vuillemin strain GHA or Metarhizium anisopliae (Metsch.) Sorokin strain F-52, under laboratory and greenhouse conditions. Nematodes and fungi were either applied alone or in combination, with nematodes added to fungi at different times. When applied alone, B. bassiana and M. anisopliae did not reduce grub numbers. Under laboratory conditions, additive interactions were found between H. megidis and B. bassiana, and between H. bacteriophora and B. bassiana or M. anisopliae in most combinations against chafer grubs; a few treatments showed synergism or antagonism. The combined effect did not differ significantly for nematode and fungal applications made simultaneously or at different times. Nematode infection and infective juveniles (IJs) production in grub carcasses were not significantly affected by the presence of a fungus. Efficacies of H. bacteriophora and M. anisopliae were affected by temperature, with grub mortality increasing at higher temperatures. Under greenhouse conditions, additive or synergistic interaction was found between H. bacteriophora and B. bassiana or M. anisopliae in different formulations in simultaneous applications or when the nematode was applied 4 weeks after the fungi, except between B. bassiana ES and H. bacteriophora. The impact of H. bacteriophora alone or in combination with M. anisopliae or B. bassiana on 3^rd instar C. lurida was comparable to that of an imidacloprid insecticide used as curative applications. More virulent fungal strains or species may be required to achieve a stronger interaction with nematodes in the management of C. lurida.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).