Abstract:Abstract: Global regulators play an important role in secondary metabolite biosynthesis and morphological development in filamentous fungi． LaeA，a key global regulator in filamentous fungi was found in 2004，regulating the expression of a variety of fungal natural product gene clusters． Besides regulation of beneficial metabolism，fungal toxin biosynthesis and morphological developmental processes，laeA also contributed to activating the expression of cryptic gene clusters and resulted in generating novel secondary metabolites． Although the mechanism of LaeA on gene activation is still unclear，it has set the stage for understanding the fungal regulatory network and discovering new natural products． This review highlight its discovery，function and regulatory mechanism in filamentous fungi．

Abstract:Abstract: Zero-valent iron ( Fe0 ) is very effective for the transformation of a wide variety of common environmental contaminants． No contaminant mineralization happened only by Fe0 corrosion，but the pollutants would be completely detoxified and degraded by the integrated microbial-Fe0 treatment，which represent a new generation of environmental remediation technologies． In this paper，the mechanism，microbial diversity and application of the integrated microbial-Fe0 treatment processes were reviewed．In addition，we also discussed the main problems and challenges in this filed．

Abstract:Abstract: Quorum sensing (QS) refers to the behavior of microorganisms to control gene expression through detection the concentration of certain signal molecules，which is correlated with cell density． In many class Ⅱ bacteriocin-producing lactic acid bacteria (LAB)，bacteriocin production is regulated by peptide pheromones via a QS mechanism． We reviewed，QS regulated class Ⅱ bacteriocin production in LAB and its regulation mechanism，components of the QS system，as well as the application of QS mechanism． The study of QS mechanism of class Ⅱ bacteriocin-producing LAB may provide a new platform for revealing the mechanism of fermentation control and regulating fermentation process． It also offers an alternative to the exploitation of food grade gene expression system．

Abstract:Abstract: ［Objective］ To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization．[Methods］Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI． Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares． Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe．［Results］ One complete plasmid was sequenced from CWBFq． pCWBFq comprised 4446 bp and had a nucleotide content of 73. 5% A + T and encoded six proteins． Protein P2，P3，P4 and P5 of pCWBFq contained 3，2，1 and 2 tranmembrane domains respectively，and their predicted signal peptide values were 0. 989，0. 505，0. 918 and 0. 914 respectively． Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9. 6% -85. 6% ，however，the homology of different SSB proteins was between 74. 0% － 89. 4% ． Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq． In addition，The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy) ，periwinkle virescence phytoplasma-Hainan stanin (PeVHn) ，chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy) ，whereas，no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj)，cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc)．［Conclusion］ The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB) ，which were for the replication of plasmid． Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains，respectively，and presumably to be localized to the membrane． The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation，among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids． CWBFq，PaWBNy，PeVHn，CWBFz andMDPy possessed distinct plasmids in terms of number and size，whereas there was no plasmid detected in JWBBj，CLYXc and BiWBNc，perhaps as a result of low homology among repA genes in plasmids of JWBBj，CLYXc and BiWBNc．

Abstract:Abstract: ［Objective］To identify the role of rmlB in synthesizing L-rhamnose in the pathogenic Escherichia coli 44277(O2:K1:H4)．［Methods］The rmlB gene was expressed and the activity of the recombinant protein was assayed by measuring the quantity of reaction product．The rmlB gene was deleted by homologous recombination，then phenotypic changes of the △rmlB mutant was analyzed by Electron Microscope，Tricine SDS-PAGE and immunological methods． Further，various methods including MALDI-TOF-MS /MS，GC-MS and NMR was used to investigate the O antigen structure of the △rmlB mutant．［Results］RmlB was confirmed to be a protein harboring the activity of dTDP-D-glucose 4，6-dehydratase through enzyme assay．The △rmlB mutant was successfully constructed and no phenotypic change was observed after compared with the wild type strain． L-rhamnose still existed in the △rmlB mutant，indicating that there may be isoenzyme of RmlB presenting in the mutant or there was a novel way synthesizing L-rhamnose in the mutant．［Conclusion］RmlB has the activity of dTDP-D-glucose 4，6-dehydratase but it is not essential for the synthesis of Lrhamnose．

Abstract:Abstract: ［Objective］ To construct and characterize a sigK gene disruption mutant of Bacillus thuringiensis and to study influence of sigK gene disruption on the activation of cry3A gene promoter.[Methods] We constructed the sigK gene disruption mutant HD△sigK by inserting kanamycin resistance gene via homologous recombination． Scanning electron microscopy and spore formation analysis were used to detect the abilities of sporulation and crystal protein formation of boththe mutant and the wild-type strain． SDS-PAGE analysis was used to detect the expression of crystal protein． Betagalactosidase assay of cry3A'-lacZ gene fusion was performed to analyze the influence of sigK gene disruption on the activation of cry3A promoter． ［Results］ The growth curve showed that mutant grew slowly in late stationary phase compared to the wild-type strain． Scanning electron microscopy and spore formation analysis indicated that no spore was produced in sigK disruption mutant． SDS-PAGE results exhibited that the expression of cry gene was significantly decreased in the mutant． Beta-galactosidase assay showed that the activation of cry3A promoter was stronger in the mutant than that in HD-73 during late stationary phase，but the disruption of sigK gene had no significant influence on the production of Cry1Ac which was initiated by cry3A gene promoter． ［Conclusion］ These results indicated that sigK gene was one of the essential genes during the sporulation of Bacillus thuringiensis，and influenced the expression of crystal protein． The expression of crystal protein which was initiated by cry3A gene promoter in sigK disruption mutant could be used to develop high-efficiency and safe biological pesticides.

Abstract:Abstract: Acidovorax citrulli(Ac) is an important bacterium that occurs in watermelon，melon and other cucurbits．It mainly damages watermelon and melon，and can cause leaf blight，fruit rot，and even mortality．［Objective］To verify the relationship between defects in the synthesis of histidine and the pathogenicity of Ac．［Methods］We generated a transposon (Tn5) mutant library on the background of strain xjl12 of Ac． Then we used subclone technology to identify the gene．［Results］The mutant could not elicit the hypersensitive response (HR) in nonhost tobacco，and its virulence was reduced． It is impaired in hisC，which encodes the protein histidinolphosphate aminotransferase． The other three genes (hisA，hisB and hisD) involved in the process of histidine synthesis were also studied． These mutants could not elicit the hypersensitive response (HR) in nonhost tobacco; their virulence was reduced significantly and disease symptoms caused by mutants were delayed for 48 hours when compared to the wild type strain． By adding exogenous histidine，pathogenicity of the mutants was restored.［Conclusion］The change of the characteristics of the mutants was directly related to the synthesis of histidine.

Abstract:Abstract: ［Objective］The genetic transformation of Valsa mali var． mali was developed by PEG-mediated protoplasts transformation．［Method］It was transformed by PEG-induced fusion of protoplasts． The plasmid pBIG2RHPH2-GFPGUS carrying hph gene was used and Valsa mali var． mali 03-8 isolate was used as the host strain．［Result］At50 mg/mL driselase + 10 mg/mL lysing enzymes concentration，the mycelium of Valsa mali var． mali cultured in YEPD medium for 48 h was hydrolyzed in 10 mL enzymes liquid /0. 5 g wet mycelium for 2 h． The protoplast yield was 4×107 CFU /mg． The transformation efficiency was 44 per g DNA． Analysis of the transformants by PCR and Southern blotting showed that the selectable marker gene hph was integrated effectively into the genome of Valsa mali var． mali．After 5 subculturing on PDA，87. 5% transformants could grow． This stability test of transformants suggested that the foreign gene hph was stable in heredity．［Conclusion］This transformation system is a valuable and important tool for the further study of the pathogenic gene of Valsa mali．

Abstract:Abstract: Methanosaeta harundinacea 6Ac displayed a cell-density dependent cell morphology transition between short rods (3μm－5μm) and filaments (＞ 200μm) ，implying a quorum sensing system in this archaeon.[Objective］This study aimed to confirm that cell morphology related quorum sensing exists in Methanosaeta harundinacea.[Methods］By using the bioassay of Agrobacterium tumefaciens NTL4，we determined the presence of acyl homoserine lactones (AHLs) in the spent cultures of Methanosaeta harundinacea 6Ac and other three methanogens．A chemical synthetic AHL，N-(β-Ketooctanoyl) -L-homoserine lactone was added into the culture of short rods to detect the morphology change.[Results］We determined that AHLs were produced by Methanosaeta harundinacea，Methanosarcina mazei，Methanothermobacterthermautotrophicus and Methanobacterium formicicum． Addition of N-(β-Ketooctanoyl) -L-homoserine lactone into theculture of Methanosaeta harundinacea 6Ac stimulated the formation of filamentous cells．［Conclusion］This study indicates that AHL-based quorum sensing may be used by several species of methanogenic Archaea．

Abstract:Abstract: ［Objective］ To detect peptide toxins in Amanita pallidorasea and to study the antifungal activities of peptide toxins against Blastomyces albicans． ［Methods］We separated and identified peptide toxins and determined its contents in the fruiting body，pileus and the mixture of stipe and volva from A． pallidorasea by HPLC and ESI-MS methods．Meanwhile，we detected antifungal activities of the crude toxin and the separated peptide toxins against Blastomyces albicans JLC31680 and JLC31681by the paper disk method．［Results］We totally got three peptide toxins: α-amanitin (α-AMA)，β-amanitin (β-AMA) and phalloidin (PHD)．The contents of α-AMA，β-AMA and PHD were 30.3 mg/g，6.99 mg/g and 9.95 mg/g in fruiting body，and 45.0 mg/g，11.1 mg/g and 11.3 mg/g in pileus．The contents of α-AMA and PHD were 11.7 mg/g and 7.98 mg/g in the mixture of stipe and volva，but the β-AMA was not detected in this part． The inhibition ratio of the crude toxin and a-AMA，β-AMA and PHD to B．albicans JLC31680 were 11.96%，32.52%，23.29%(P＜0.01) and 15.46%(P＜0.05)．The inhibition ratio of the crude toxin and β-AMA to B．albicans JLC31681 was 10.16% and 11.10%(P＜0.01)，while that of α-AMA s was 6.89%(P＜0.05)．[Conclusions] A．pallidorasea is a new resource of peptide toxins with antifungal activity．

Abstract:Abstract: ［Objective］ Aflatoxin B1(AFB1)is extremely mutagenic，toxic and a potent carcinogen both to humans and livestock． Aflatoxin-oxidase (AFO)was an aflatoxin-converting enzyme previously purified by us from Armillaria tabescens．In order to know better about the molecular characterization of this distinct enzyme，we expressed，purified and characterized the His6 tag fused aflatoxin-oxidase．［Methods］Based on sequences of peptides fragments of AFO previously obtained by Electrophoresis-Electrospray Ionization tandem mass spectrometry (ESI-MS/MS)，we cloned the cDNA of AFO using Switching Mechanism At 5' end of the RNA Transcript (SMART) Rapid Amplification of cDNA Ends (RACE) technology and expressed this gene as a fusion protein in Pichia pastoris by using pPIC9-afo as vector．We purified the fusion enzyme using nickel affinity chromatography．We identified the recombinant aflatoxin-oxidase (rAFO) by both western blot and peptide mass fingerprinting (PMF)．Moreover，we characterized several enzymatic properties of the rAFO using AFB1 as the substrate including Km value，optimum temperature，optimum pH，thermal stability and pH stability．[Results] The AFO gene is 2321 bp long with a coding region of 2088 bp encoding 695 amino acids．Peptide mass fingerprinting (PMF) identification showed a 63.2% coverage of the molecule compared to the theoretical tryptic cleavage of the rAFO．The recombinant aflatoxin oxidase was purified 5. 99-folds using nickel affinity chromatography．It has a specific activity of 234 U/mg． Kinetics studies showed that the rAFO converted AFB1 with the Km value of 3.93±0.20×10－6 mol /L under its optimal conditions of pH6.0 and 30℃．Thermostability investigation revealed that the rAFO had a half-life of 90 min at 30℃，and pH stability results suggested that the rAFO was relatively stable when pH ranged from 5.5 to 7.5．[Conclusion] It appears to be the first successful production of the recombinant aflatoxin oxidase (rAFO) with AFB1-converting ability from Armillaria tabescens．The purified rAFO with preferably AFB1-converting activity confirms that this recombinant aflatoxin oxidase is now ready for further studying．

Abstract:Abstract: ［Objective］In order to explore the diversity of cultured halophilic archaeon from hypersaline environments in Lop Nur region and their potential application． Methods］ Total 13 soil samples were collected from Lop Nur regions．Halophilic archaea strains were isolated and identified by 16S rRNA gene sequence analysis．In addition，17 strains were selected based on different branches in pylogenetic tree，and their salt concentration tolerance and amylase，protease，esterase activities were further detected by conventional methods．［Results］The 16S rRNA gene sequences of 56 selected strains were determined，and the phylogenetic analysis was carried out．These strains were classified into 10 known genera and 5 new potential genera，and the Shannon index was 1.820．The range of salt concentration tolerance of most strains was 10%－35% (optimum at 20%－25%)．Amylase positive rate was 70.6%，protease positive rate was 35.3% and esterase positive rate was 82.4%．［Conclusion］Diverse halophilic archaeon were discovered in Lop Nur regions．The isolation methods that we used were successful for isolating halophilic archaeon from these areas，which provided the technical basis to future explore the resources of halophilic archaeon in Lop Nur regions．

Abstract:Abstract: [Objective］In order to find new strains to degrade fomesafen in contaminated soil，we isolated and identified a high-efficiency degrading bacterium from polluted soil． The degrading characteristics and remediation ability of the strain were also studied．［Methods］Characteristics of morphological，physiological，biochemical and 16S rRNA sequence were applied to identify the strain．The optimum growth conditions were obtained by studying the effect of environmental factors such as fomesafen concentration，primary pH and temperature on the strain． The strains remediation ability to fomesafen-polluted soil was verified by sensitive crop and target weeds bioassay in pot soil．［Results］A high-efficiency degrading strain FB8 that used fomesafen as sole carbon source was isolated from soybean field suffering fomesafen in Heilongjiang province．It was initially identified as a member of the genus Pseudomonas．The strain could degrade 86.75% of 500mg /L fomesafen within 96 h．Its optimal growth conditions were determined as follows: 500mg /L fomesafen，primary pH between 6.0 and 8.0，and at 35 to 37℃. The strain could remedy the sensitive crop maize and sorghum biomass after treating for 30 d for soil contaminated with 5 mg / kg of fomesafen． [Conclusion] A fomesafendegrading strain FB8 was selected from fomesafen-contaminated soil in Heilongjiang Province．The strain was closely related to Pseudomonas mendocina． The strain was a suitable candidate for bioremediation of fomesafen-contaminated soil．

Abstract:Abstract: ［Objective］Postharvest decay resulted from anthracnose caused by pathogens Colletotrichum gloeosporioides and blight diseases caused by Phytophthora nicotianae leads to significant loss of papaya fruits． In order to reduce such loss，we isolated endophytic bacteria that may possess powerful antagonistic activities toward these pathogens for effective biological control of anthracnose and blight diseases． Methods］The methods of dilution and inhibition circle were used for isolating and screening endophytic bacteria from papaya fruit． Based on morphological，physiological and biochemical characteristics，and homology analysis of the partial sequence of 16S rDNA，an endophytic bacterium was identified．The colonization of the antagonistic endophyte in papaya was detected by inoculating suspension of strains in caudices of papaya plant after Rifampicin-resistant mutants (rifr) induction．The effects on diseases caused by Colletotrichum gloeosporioides and Phytophthora nicotianae were tested by preharvest and postharvest experiments．[Results] One of the endophytic bacteria named MGP3 was selected from the papaya pericarp and identified as Pseudomonas aeruginosa ( Accession No．JF708186)．This bacterium was able to colonize in the laminae，leafstalk or pericarp of papaya，and strongly inhibit 10 phytopathogens． In the postharvest experiment，MGP3 inhibited 50% anthracnose and 71% blight of harvested papaya fruits．The application of MGP3 at five preharvest stages of papaya significantly reduced latent infection rate of Colletotrichum gloeosporioides and disease index of anthracnose．［Conclusion］Antagonistic endophytic bacterium MGP3 isolated from papaya fruit had potential application value as a biological control agent．

Abstract:Abstract: [Objective] To accelerate the conversion of rice straw into feeds in the low-temperature region，a microbial community was constructed by continuous enrichment cultivation． Microbial diversity and dynamics during the fermentation at 10℃ was analyzed．［Methods］The community was selected at 5℃ under static condition．To analyze the inoculating effects，the community and commercial inoculant (CI:composed of Lactobacillus plantarum，Enterococcus faecium，L.salivarilus，Pediococcus acidilactici) were respectively inoculated into the rice straw for 30 d fermentation at 10℃．Fermented products were detected by gas chromatography–mass spectrometry (GC-MS)．Composition microorganisms of the community were analyzed using cloning library．Microbial dynamics during the fermentation was detected by denatured gradient gel eletrophoresis (DGGE)．Quantitative PCR was used for tracking the composition microorganisms of the community during the fermentation．［Results］The results from 16S rDNA cloning library showed that the community was mainly composed of Lactobacillus spp. and Leuconostoc spp．At 6d fermentation，the pH and the lactic acid bacterial colony forming units (LAB CFUs) in the fermented rice straw with the community amounted to 4.3 and 2.9×109 CFU/g fresh matter (FM)，respectively．The pH and LAB CFUs with the CI were respectively 5.3 and 2.9×109 CFU/g FM．At 30 d fermentation，the lactic acid concentrations with the community and the CI were respectively 8. 1g / kg FM and 2.0g/kg FM． From DGGE patterns， both L． sakei and Leuconostoc inhae of the community were detected at 6d fermentation and existed during the fermentation． For the treatment with the CI，the uncultured bacterium was detected at 6d fermentation besides the composition microorganisms of the CI． At 16d and 30d fermentation，only L． plantarum and E． faecium were detected． Quantitative PCR showed DNA mass of L． sakei amounted to 41. 0% at 6d fermentation in the treatment with the community． At 16d，DNA mass of L． sakei was 65% ． The highest value (5.5% ) of DNA mass of Le inhae appeared at 6d of fermentation ［Conclusion］ The community could effectively colonize into the rice straw fermentation system and accelerate the fermentation process at low temperature． The dominating microorganism of the community was L． sakei at 10℃ ．

Abstract:Abstract: ［Objective］To investigate the role of ompR gene from Salmonella enteritidis in biofilm formation and virulence．［Methods］We constructed an ompR mutant of Salmonella enteritidis by suicide plasmid pGMB151． Biofilm forming ability of the mutant was detected by crystal violet assay and scanning electron micrography． Virulence of the mutant was determined by assay of adherence to and invasion of epithelial cells，and mouse challenge experiments． ［Results］ The ompR mutant was confirmed by RT-PCR and the pattern of outer membrane protein． The mutant did not produce cellulose， curli，and biofilm，and showed similar adherence percentage to and invasion percentage of epithelial cells as wild type strain． In addition，intraperitoneal challenge of bacteria in BALB / c mice revealed that LD50 of the mutant strain was 106. 67 CFU，while that of the wild type strain was less than 2 CFU． ［Conclusion］ These data indicate that the ompR gene is involved in both biofilm formation and virulence in Salmonella enteritidis．

Abstract:Abstract: ［Objective］To develop cryopreservation for Oenococcus oeni． ［Methods］We investigated the influence of the age and concentration of the strain in the protective medium，rate of freezing，temperature of thawing and the protective medium on the number of viable cells after cryopreservation by colony counting． ［Results］We got the highest number of viable cells after cryopreservation by collecting cells in the early stationary growth phase，keeping their concentration in the protective medium at 109 CFU /mL，transferring cells into liquid nitrogen directly and thawing in a water bath at 37℃ ． The ratio of viable cells was above 99% for 21 of the Oenococcus oeni after storing in liquid nitrogen for 6 months．［Conclusion］ We The storage of Oenococcus oeni in liquid nitrogen is convenient for strains preserved in culture collections．

Abstract:Abstract: ［Objective］The study was carried out to construct and characterize Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae and to test its immunogenicity in mice． ［Method］We made p36，p46，p65 and p97R1-Nrdf，the main immunogenic genes of Mycoplasma hyopneumoniae，to insert into the prokaryotic expression plasmid pYA3493. Then these recombinant plasmids and pYA3493 were electroporated into C500 asd- mutant，resulting in the recombinant Salmonella choleraesuis vaccine strains C36( pYA-36) ，C46( pYA-46) ，C65( pYA-65) ，C97R1-Nrdf( pYA-97R1-Nrdf) and CpYA( pYA3493) ． We characterized these recombinant Salmonella choleraesuis vaccine strains and tested the immunogenicity in mice by intramuscular injection or orally immunized． ［Result］ The results of the immunogenicity in mice indicated that the group orally immunized with C36，C46，C65，C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36，C46，C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ( P ＜ 0. 01 ) ． The group intramuscular injected with C36，C46，C65 showed higher IFN-γ production than the group injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ( P ＜ 0. 05) ，but there was no significant difference between the group orally immunized with C36，C46，C65 and the group orally immunized with C36，C46，C65，C97R1-Nrdf ( P ＞ 0. 05) ． The highest level of IL-4 was found in the group orally immunized with C36，C46，C65; higher levels of IL-4 was observed in the group orally immunized with C36，C46，C65，C97R1-Nrdf than the group injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ; and the lowest IL-4 level was found in the group injected with C36，C46，C65. There were no significant differences among them ( P ＞ 0. 05) ． The Mycoplasma pneumoniae antibody，IFN-γ or IL-4 production of the each group was obviously higher than the control group ( P ＜ 0. 01) ． ［Conclusion］The attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine．

Abstract:Abstract: ［Objective］Chlorine dioxide ( ClO2) is a highly effective disinfectant for food and potable water treatment．However，knowledge on its action mechanism remains unexplored． The present study aims to determine the role of respiratory inhibition in the bactericidal effects of ClO2.[Methods] Transmission electron microscopy was used to observe the ultra structural alteration of the mitochondrion． Fluorescence-based flow cytometry analysis was employed to determine the disruption of mitochondrion membrane potential． Respiratory inhibition was detected by measuring the oxygen consumption． The results obtained were compared with those of plate counting． ［Results］ No visible physiological alteration in the shapes and structures of the mitochondria was found． The rate of collapse in mitochondrial membrane potential increased with the death rate，but the respiratory inhibition rates were always significantly lower than the death rates． The death rates detected by the aerobic and anaerobic methods did not differ significantly． ［Conclusion］ ClO2-induced damages to the mitochondria were positive correlated with the death rates，but respiratory inhibition was not the primary target site for cell killing．