Biological control of Botrytis cinerea and plant growth promotion potential by Penicillium citrinum in chickpea (Cicer arietinum L.)

A total of 48 fungi were characterised for their antagonistic potential against Botrytis cinerea causing Botrytis Gray Mold (BGM) disease in chickpea by dual culture and metabolite production assays. The culture filtrate of the most promising isolate, VFI-51, was purified by various chromatographic techniques and identified as ‘citrinin’ by nuclear magnetic resonance and mass spectrometry studies. The efficacy of citrinin was demonstrated to control BGM in chickpea under greenhouse conditions. The sequences of 18S rDNA gene of the VFI-51 matched with Penicillium citrinum in BLAST analysis. The VFI-51 produced siderophore, hydrocyanic acid, indole-3-acetic acid, lipase, protease and β-1,3-glucanase; grew well in NaCl (up to 15%), at pH between 7 and 11 and temperatures between 20°C and 40°C; and was compatible with fungicides bavistin and thiram. Under greenhouse and field conditions, VFI-51 significantly enhanced the nodule number, nodule weight, root and shoot weight and stover and grain yield over the un-inoculated control. In the rhizosphere, VFI-51 also significantly enhanced total N, available P and OC over the un-inoculated control. Scanning electron microscopy analysis revealed that VFI-51 colonised on the roots of chickpea. This study concluded that VFI-51 has the potential for biocontrol of BGM and plant growth promotion in chickpea.     

Degradation of organophosphonates by Penicillium citrinum

Utilization of organophosphonates as the sole source of phosphorus, carbon or nitrogen by a soil isolate of Penicillium citrinum was studied. Penicillium citrinum was found to utilize 2-aminoethylphosphonic and 2-oxoalkylphosphonic acids as the sole phosphorus source whereas 1-hydroxyalkylphosphonates as well as 1-aminoalkylphosphonates and their dipeptides did not support the growth of the fungus. The mould did not metabolize any of the phosphonates tested, when they served as the sole carbon or nitrogen source.
Penicillium citrinum is perhaps the first mould strain isolated from soil, shown to be capable of organophosphonate degradation.     

Secondary metabolites characteristic of Penicillium citrinum, Penicillium steckii and related species

Two new carboxylic acids, tanzawaic acid E (1) and F (2) in addition to the unknown benzopyran 3,7-dimethyl-1,8-dihydroxy-6-methoxy-isochroman (3), and the known mycotoxin 3,7-dimethyl-8-hydroxy-6-methoxyisochroman (4) were produced by a marine-derived strain of Penicillium steckii isolated from an unidentified tunicate. The carboxylic acids and the benzopyran were identified on the basis of mass spectrometry, and one and two dimensional NMR spectroscopic techniques. The structures 1 and 2 resemble tanzawaic acid A-D, previously isolated from Penicillium citrinum. Screening of isolates of species related to P. citrinum and P. steckii showed that P. citrinum (25 isolates) consistently produced citrinin and tanzawaic acid A, P. steckii (18 isolates) produced isochroman toxins (except 2) and tanzawaic acid E, P. sizovae consistently produced tanzawaic acid A, P. corylophilum (10 isolates) produced citreoisocoumarinol and P. sumatrense (15 isolates) always produced curvularin.     

Specificity of a Nuclease from Penicillium citrinum

The enzyme with high milk clotting activity produced by Irpex lacteus was partially purified by a CM-cellulose chromatography. Throughout the over-all process, the enzyme was purified approximately 9-fold from a crude powder with about 22.8% recovery of the original activity. The MCA/PU ratio of this fraction was 2.51 and the specific milk clotting activity was 188.7.

The purified enzyme is a sort of acid protease with optimum pH of 2.5 for casein digestion and 4.0 for hemoglobin digestion. The Lineweaver-Burk plot, when casein was used as a substrate, showed that the Km value of the enzyme was about 0.07% and the V_max value was 0.4. The molecular weight of the enzyme is about 34,000, the isoelectric point is pH 5.2 and a ultraviolet absorption maximum is at 277 mμ. The enzyme has not yet been crystalized but seems to be a sort of glycoprotein, because the Molish reaction was positive at the present purification stage.

Some enzymological properties of the enzyme was studied and compared with those of a calf rennet and Mucor rennet. In some respects such as pH optima, pH stability, thermostability and temperature optima, the enzyme is Mucor rennet alike. On the other hand, as to the increase in activity along with decrease in pH of milk and the increase in activity along with the addition of Ca ion, the enzyme is not very different from the calf rennet. However, proteolysis of milk casein by the enzyme was fairly higher than by the calf rennet.

As to the production of enzymes, I. lacteus can produce at least three types of proteases into liquid media. When, for example, R medium was used, only one type of protease, that is the fraction A, could mainly be produced and it was this enzyme that assumed to be a rennet like enzyme.     

Purification of a Nuclease from Penicillium citrinum

A nuclease was purified about 1500-fold with a recovery of 20% from an aqueous extract of culture of a pigmentless mutant VI–10–14 of Penicillium citrinum on wheat bran. The purified preparation was homogeneous on the basis of the criteria of ultracentrifugation and disc gel electrophoresis. The preparation was essentially free of 5′-nucleotidase, non-specific phosphomonoesterase, non-specific phosphodiesterase and 3′-monoester forming nuclease. The preparation hydrolyzed phosphodiester bonds in RNA and DNA to yield 5′-mononucleotides, and also the phosphomonoester bond in 2′- and 3′-AMP to yield nucleoside and inorganic phosphate. The enzyme activities toward these substrates were not separated and relative ratio of their specific activities remained constant throughout the purification, suggesting that a single enzyme was responsible for these activities.     

Effect of water activity and temperature on growth of Penicillium citreoviride and Penicillium citrinum on MiGao (rice cake)

Contamination by mold is a serious problem in steam-cooked rice cake, a traditional Chinese food. Growth responses to different temperatures and water activity values for Penicillium citreoviride and Penicillium citrinum, two of the most common molds, were investigated. Partial least square regression analysis showed that the growth of the two fungi did not differ in response to changes in water activity and temperature. Optimum water activity for growth was 0.90 and optimum temperatures for growth were 30 degrees C in most cases.     

Purification and Properties of Two Galactanases from Penicillium citrinum

Two galactanases (I and II) were purified to homogeneous states from water extracts of a wheat bran culture of Penicillium citrinum. Although these enzymes were separable by affinity chromatography and distinct on Polyacrylamide gel electrophoresis, they were similar in physical and enzymatic nature. They had almost the same molecular weight (3.5 } 10⁴) and isoelectric point (pH 4.2), and similar amino acid compositions and carbohydrate contents (3%). Alanine and leucine were detected for both enzymes as the N- and C-terminal amino acids, respectively.

These enzymes were most active at pH 4.5 and 55%C, and were stable between pH 4 and 10 (at 15%C for 24hr), and below 55%C (at pH 5.5 for 10min). The enzymes hydrolyzed soybean arabinogalactan to produce galactose and several galac-tooligosaccharides, but did not attack coffee bean arabinogalactan. Therefore, these enzymes were suggested to be endo-l,4-²-D-galactanases.

The enzymes also attacked ONPG and PNPG, and lag phases were observed at the beginning of the reactions.

Among the compounds examined, Hg²⁺ and Fe³⁺ inhibited the enzyme actions. ONPG-hydrolyzing activities of the enzymes were inhibited by some sugars such as lactose, galactose, arabinose, glucose and xylose.

Galactobiose, -triose and -tetraose prepared from soybean arabinogalactan with purified galactanase I were found to be further hydrolyzed by the enzymes. A lag phase was also observed in the time course of hydrolysis of galactobiose.     

Double-Stranded Ribonucleic Acid in Viruses of Penicillium citrinum

[This corrects the article on p. 811 in vol. 34.].     

Presence of Two Virus-Like Particles in Penicillium citrinum

Two icosahedral virus-like particles (28 and 19 nm in diameter, respectively) have been detected in sporogenic and asporogenic segregants of a strain of Penicillium citrinum. The distribution of the two particles differed among the two segregants.     

来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PcFAE)并进行了酶学性质的研究,初步探讨了PcFAE对麦糟的酶解作用。【方法】利用(NH4)2SO4沉淀、DEAE-Sepharose Fast Flow离子交换层析、Phenyl Sepharose6Fast Flow疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kDa,全酶分子量约为58kDa。其最适pH为6.0,最适温度为45℃-65℃,在pH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、Fe2+、Mn2+、Ca2+和Na+对酶活有一定的促进作用,Zn2+对PcFAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EDTA对PcFAE活性无明显影响。PcFAE的kcat/Km对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PcFAE对MpCA的催化效率最高。PcFAE作用于麦糟,当5U PcFAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。     

Citrinolactones A, B and C, and Sclerotinin C, plant growth regulators from Penicillium citrinum

New plant growth regulators, named citrinolactones A (1), B (2) and C (3) and sclerotinin C (4), were isolated from Penicillium citrinum and their structures established by spectroscopic methods including 2D NMR. Compounds 1 and 4 increased root growth in proportion to their concentration from 3 to 300 mg/l. In contrast, 2 completely inhibited root growth at a concentration of 300 mg/l and 3 did not show any effect on root growth in a concentration range of 3-300 mg/l.     

Double-stranded ribonucleic acid in viruses of Penicillium citrinum.

The nucleic acid content of two viral populations in a strain of Penicillium citrinum is shown to be double-stranded ribonucleic acid, resolved through polyacrylamide gel electrophoresis into 10 size groups ranging from 1.17 to 3.98 megadaltons.     

Primary structure of nuclease P1 from Penicillium citrinum

The primary structure of nuclease P1, which cleaves both RNA and single-stranded DNA, from Penicillium citrinum was elucidated. The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter protease I of the reduced and S-aminoethylated protein and by digestion with Staphylococcus aureus V8 protease of the reduced and S-carboxymethylated protein. Four half-cystine residues were assigned to Cys72-Cys217 and Cys80-Cys85. N-Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197. Fast-atom-bombardment and laser-ionization MS were successfully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively. Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease S1 from Aspergillus oryzae. The His-Phe-Xaa-Asp-Ala sequence (positions 60-64) is similar to the sequence (His-Phe-Asp-Ala) involving the active-site His119 of bovine pancreatic RNase A, and the Pro-Leu-His sequence (positions 124-126) is identical with the sequence involving the active-site His134 of porcine pancreatic DNase I.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

Double-stranded ribonucleic acid in viruses of Penicillium citrinum.

The nucleic acid content of two viral populations in a strain of Penicillium citrinum is shown to be double-stranded ribonucleic acid, resolved through polyacrylamide gel electrophoresis into 10 size groups ranging from 1.17 to 3.98 megadaltons.     

Purification of a Penicillium citrinum lipase by chromatographic processes

A lipase from a wild strain of Penicillium citrinum was purified by ammonium sulphate precipitation, gel filtration chromatography on a Superose 6 column and hydrophobic interaction chromatography (HIC) on a Phenyl Superose column. The yield and purification factor were 15.2% and 379 fold, respectively. The gel filtration step was efficiently scaled-up in a Superose 6 preparative grade column and after this step, the lipase was recovered in the form of a high molecular weight aggregate. The partial disaggregation of the complex was achieved by HIC and elution with 1.0% (w/v) CHAPS. The lipase produced by Penicillium citrinum forms a dimmer of 63?000 Da, as determined by SDS-PAGE, and it accumulates in the fermentation broth as high molecular weight aggregates (>2?00?000 Da). The analysis of the dimmer showed two subunits with similar molecular weights (31?000–33?000 Da) and isoelectric points (4.8–5.0).     

Inhibition of growth of Sclerotinia minor and other pathogens by citrinin in the filtrate of Penicillium citrinum

Penicillium citrinum Thom, isolated from the sclerotia of Sderotinia minor, was cultured in a broth of Czapek-Dox for 4 to 8 weeks. The filtrate obtained was incorporated into potato dextrose agar or Czapek-Dox agar at different concentrations (v/v). The amended media were tested for mycelial growth of S. minor and other pathogens. Mycelial growth of S. minor was completely inhibited on media amended with 20% (v/v) filtrate of P. citrinum, and considerable inhibition of S. minor occurred at 10 and 15% concentrations. Mycelial growth of S. major, Sclerotium rolfsii, Rhizoctonia solani (AG-4) was inhibited by similar concentrations of filtrate of P. citrinum. Inhibitor(s) in the filtrate were extracted with ethyl acetate and tentatively identified as citrinin. Citrinin was shown to be an active component in the filtrate against mycelial growth of S. minor, S. major and Sclerotium rolfsii.Cooperative investigation of U.S. Department of Agriculture, Agriculture Research Service and Oklahoma State University. Journal Article No. 4989, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA or by Oklahoma State University, or imply their approval to the exclusion of other products or vendors that may also be suitable.