Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformat...Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.展开更多
Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but...Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but the frequencies are often very low. In the present study, we replaced the Ku80 homolog （a gene of the non-homologous end-joining （NHEJ） pathway） with the hygromycin resistance cassette （hph） by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in KuSO-deficient strain （△dKuSO） of P. digitatum. However, using △pdKuSO as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous inte- gration and the created △PdKuSO strain would be a good candidate for rapid gene function analysis in P. digitatum.展开更多
Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, se...Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, sequence, predicted construction, base bias, expression levels and potential targets were determined as well. Through pathway and KEGG enrichment analysis, the miRNA target genes were mostly involved in carbohydrate metabolism, transport and catabolism, translation and amino acid metabolism. The target genes involved in aflatoxin biosynthesis and proteasome had a higher rich factor value. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of P. digitatum at the transcriptional level.展开更多
Growth curves fitted Penicillium digitatum were used to analyze the effect of temperature, pH and aw on their growth. To asses observance of independent variable effects on all growth parameters (λ, latency time, μ,...Growth curves fitted Penicillium digitatum were used to analyze the effect of temperature, pH and aw on their growth. To asses observance of independent variable effects on all growth parameters (λ, latency time, μ, growth rate and ymax maximal growth) a slow growth strain of P. digitatum was employed. Growth curves were obtained at different conditions of temperature (10?C - 40?C), pH (3.0 - 7.0) and water activity (0.800 - 0.990) and growth parameters were calculated by fitting of logistic model. Polynomial models were made by linear regression and all terms (linear, quadratic and interactive effects) were statistically significant (p < 0.001). All growth parameters, including maximal growth were affected by environmental conditions;pH effect was more important on maximal growth than that on lag time or growth rate. In some aspects results and modeling behavior of P. digitatum, are very similar to modeling of bacterial growth.展开更多
In order to investigate whether an air–water plasma jet is beneficial to improve the efficiency of inactivation, a series of experiments were done using a ring-needle plasma jet. The water content in the working gas...In order to investigate whether an air–water plasma jet is beneficial to improve the efficiency of inactivation, a series of experiments were done using a ring-needle plasma jet. The water content in the working gas（air） was accurately measured based on the Karl Fischer method. The effects of water on the production of OH（A;Σ;–X;Π;） and O（3p;P–3s;S） were also studied by optical emission spectroscopy. The results show that the water content is in the range of 2.53–9.58 mg l;, depending on the gas/water mixture ratio. The production of OH（A;Σ;–X;Π;） rises with the increase of water content, whereas the O（3p;P–3s;S） shows a declining tendency with higher water content. The sterilization experiments indicate that this air–water plasma jet inactivates the P. digitatum spores very effectively and its efficiency rises with the increase of the water content. It is possible that OH（A;Σ;–X;Π;） is a more effective species in inactivation than O（3p;P–3s;S） and the water content benefit the spore germination inhibition through rising the OH（A;Σ;–X;Π;） production. The maximum of the inactivation efficacy is up to 93% when the applied voltage is -6.75 kV and the water content is 9.58 mg l;.展开更多
Cyphostemma digitatum （Vitaceae） is a perennial, climbing, succulent undershrub with compound fleshy leaves and tendrils. The plant is used mainly as a food flavoring, but it is also a main constituent of traditiona...Cyphostemma digitatum （Vitaceae） is a perennial, climbing, succulent undershrub with compound fleshy leaves and tendrils. The plant is used mainly as a food flavoring, but it is also a main constituent of traditional Yemeni soup （Marak）. Besides that, it has been described to be used as a medicinal plant. The aim of this work was to study the hepatoprotective effect of the aqueous extract of C. digitatum against CCl4-induced liver injury in Guinea pigs. Animals were divided into four groups. Group I, served as normal control. Group II received 2 mL CCl4/kg b.w. diluted with olive oil, at 1：1 ratio on day 11. Group III （test group） was pre-treated orally with 100 mg/kg b.w. aqueous leaves extract of C. digitatum for 10 days followed by subcutaneous injection of CC14 （2 mL/kg b.w.）, once on day 11. Group IV were orally given Liv-52 （100 mg/kg b.w.） once daily for 10 days followed by subcutaneous injection of CC14. Our results show that, the activity of serum hepatic enzymes （alanine aminotranferase （ALT）, aspartate aminotranferase （AST）, and alkaline phosphatase （ALP）） were significantly elevated in Guinea pigs treated with CCl4, while both the C. digitatum extract and Liv-52 reduced significantly these enzymes activity. Also, the levels of glucose, urea, cholesterol and triglycerides were decreased when compared with intoxicated control. Histopathological examination of intoxicated animals showed fatty changes, inflammation and necrosis indicating liver damage, while the animals received C. digitatum or Liv-52 showed less pathological effects or normal liver when compared to animals treated with CC14 alone. Biochemical and histological results confirm the hepatoprotective effect of aqueous extract of C. digitatum.展开更多
基金the National Natural Science Foundation of China (No. 30571236)the Modern Agriculture (Citrus) Technology System (MATS) of Chinathe Science and Technology Department of Zhejiang Province, China (No. 2007C22007)
文摘Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.
基金supported by the National Natural Science Foundation of China(Nos.31371961 and 31071649)the China Agriculture Research System(No.CARS-27)the Special Fund for Agro-scientific Research in the Public Interest(No.201203034),China
文摘Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but the frequencies are often very low. In the present study, we replaced the Ku80 homolog （a gene of the non-homologous end-joining （NHEJ） pathway） with the hygromycin resistance cassette （hph） by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in KuSO-deficient strain （△dKuSO） of P. digitatum. However, using △pdKuSO as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous inte- gration and the created △PdKuSO strain would be a good candidate for rapid gene function analysis in P. digitatum.
文摘Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, sequence, predicted construction, base bias, expression levels and potential targets were determined as well. Through pathway and KEGG enrichment analysis, the miRNA target genes were mostly involved in carbohydrate metabolism, transport and catabolism, translation and amino acid metabolism. The target genes involved in aflatoxin biosynthesis and proteasome had a higher rich factor value. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of P. digitatum at the transcriptional level.
文摘Growth curves fitted Penicillium digitatum were used to analyze the effect of temperature, pH and aw on their growth. To asses observance of independent variable effects on all growth parameters (λ, latency time, μ, growth rate and ymax maximal growth) a slow growth strain of P. digitatum was employed. Growth curves were obtained at different conditions of temperature (10?C - 40?C), pH (3.0 - 7.0) and water activity (0.800 - 0.990) and growth parameters were calculated by fitting of logistic model. Polynomial models were made by linear regression and all terms (linear, quadratic and interactive effects) were statistically significant (p < 0.001). All growth parameters, including maximal growth were affected by environmental conditions;pH effect was more important on maximal growth than that on lag time or growth rate. In some aspects results and modeling behavior of P. digitatum, are very similar to modeling of bacterial growth.
基金supported by National Natural Science Foundation of China (NSFC) under Grants No. 51407020National Key Technology Research and Development Program of the Ministry of Science and Technology of China under Grants No. 2014BAC13B05Visiting Scholarship of State Key Laboratory of Power Transmission Equipment & System Security and New Technology (Chongqing University) No. 2007DA10512716404
文摘In order to investigate whether an air–water plasma jet is beneficial to improve the efficiency of inactivation, a series of experiments were done using a ring-needle plasma jet. The water content in the working gas（air） was accurately measured based on the Karl Fischer method. The effects of water on the production of OH（A;Σ;–X;Π;） and O（3p;P–3s;S） were also studied by optical emission spectroscopy. The results show that the water content is in the range of 2.53–9.58 mg l;, depending on the gas/water mixture ratio. The production of OH（A;Σ;–X;Π;） rises with the increase of water content, whereas the O（3p;P–3s;S） shows a declining tendency with higher water content. The sterilization experiments indicate that this air–water plasma jet inactivates the P. digitatum spores very effectively and its efficiency rises with the increase of the water content. It is possible that OH（A;Σ;–X;Π;） is a more effective species in inactivation than O（3p;P–3s;S） and the water content benefit the spore germination inhibition through rising the OH（A;Σ;–X;Π;） production. The maximum of the inactivation efficacy is up to 93% when the applied voltage is -6.75 kV and the water content is 9.58 mg l;.
文摘Cyphostemma digitatum （Vitaceae） is a perennial, climbing, succulent undershrub with compound fleshy leaves and tendrils. The plant is used mainly as a food flavoring, but it is also a main constituent of traditional Yemeni soup （Marak）. Besides that, it has been described to be used as a medicinal plant. The aim of this work was to study the hepatoprotective effect of the aqueous extract of C. digitatum against CCl4-induced liver injury in Guinea pigs. Animals were divided into four groups. Group I, served as normal control. Group II received 2 mL CCl4/kg b.w. diluted with olive oil, at 1：1 ratio on day 11. Group III （test group） was pre-treated orally with 100 mg/kg b.w. aqueous leaves extract of C. digitatum for 10 days followed by subcutaneous injection of CC14 （2 mL/kg b.w.）, once on day 11. Group IV were orally given Liv-52 （100 mg/kg b.w.） once daily for 10 days followed by subcutaneous injection of CC14. Our results show that, the activity of serum hepatic enzymes （alanine aminotranferase （ALT）, aspartate aminotranferase （AST）, and alkaline phosphatase （ALP）） were significantly elevated in Guinea pigs treated with CCl4, while both the C. digitatum extract and Liv-52 reduced significantly these enzymes activity. Also, the levels of glucose, urea, cholesterol and triglycerides were decreased when compared with intoxicated control. Histopathological examination of intoxicated animals showed fatty changes, inflammation and necrosis indicating liver damage, while the animals received C. digitatum or Liv-52 showed less pathological effects or normal liver when compared to animals treated with CC14 alone. Biochemical and histological results confirm the hepatoprotective effect of aqueous extract of C. digitatum.
