Structure of Colletochlorin from Colletotrichum nicotianae

Pseudomonas melanogenum ATCC 17806 required methionine, cysteine, cystine, cystathionine, homocysteine or homocystine for growth. However, the addition of these amino acids decreased remarkably l-DOPA (3,4-dihydroxyphenyl-l-alanine) production by the bacterium. l-DOPA production by the bacterium was further affected by the amount of the substrate, the method of its addition and by the addition of antioxidants, as was the case with Vibrio tyrosinaticus.

Under suitable conditions about 8 mg/ml of l-DOPA were produced from 8.6 mg/ml of l-tyrosine.     

Comparison of Suppressiveness of Vermicomposts Produced from Animal Manures and Sewage Sludge against Phytophthora nicotianae Breda de Haan var. nicotianae

The degrees of suppression produced by vermicomposts produced from cattle manure, sheep manure or horse manure and by vermicomposts produced from sewage sludge were compared in greenhouse experiments. The effect of these vermicomposts on the growth and infection of tomato seedlings by Phytophthora nicotianae var. nicotianae was studied. The density of the pathogen and the number of micro-organisms in container media amended with vermicomposts were also analysed. The vermicomposts produced from animal manure significantly reduced the infection of tomato seedlings by the pathogen. The density of P. nicotianae in media which included these vermicomposts was similar to that in infested peat substrate (control treatment). The vermicomposts from sewage sludge did not protect tomato seedlings against P. nicotianae . They also significantly inhibited growth of the plants as well as decreasing the density of the pathogen in container media. In general the vermicomposts had no effect on total number of micro-organisms in potting media compared with control. They only had higher levels of actinomycetes but this did not appear to correspond with their ability to suppress the pathogen.     

Structure of Colletochlorin D from Colletotrichum nicotianae

Complete isolation of Fraction 1 protein from alfalfa leaves was achieved by a combination of ammonium sulfate fractionation and gel filtration. Analytical ultracentrifugation gave a S₂₀°,w value of 18.0. Judging from the CD spectrum the protein contains a large amount of β-form as well as tobacco F-l protein. Electron micrographs showed closely similar appearances for the two F-l proteins. The F-l protein (from alfalfa leaves) was separated to large subunits (53,000 daltons) and small subunits (14,000 daltons) on SDS gel electrophoresis. Further the amino acid composition of the large subunit was found similar to those of tobacco and spinach, but considerably different from them in small subunits.     

Microassay for biological and chemical control of infection of tobacco byPhytophthora parasitica var.nicotianae

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Pathogenicity of Rhizoctonia solani and Phytophthora nicotianae to Brassicaceae cover crops

Soil-borne diseases can reduce nursery crop performance and increase costs to nursery producers. In particular, soil-borne diseases caused by Phytophthora nicotianae and Rhizoctonia solani are the most economically important problems of Southeastern United States nursery producers. Methyl bromide was widely used as a standard treatment for management of soil-borne diseases until the implementation of the Montreal protocol. Since then, many chemical and non-chemical soil-borne disease management methods have been tested, but are not yet providing effective and consistent results like methyl bromide. Cover crops that belonged to the Brassicaceae family can be incorporated into the soil to control soil-borne diseases and this process is widely known as biofumigation. But, the use of Brassicaceae cover crops has not been widely explored as a method of controlling soil-borne diseases in woody ornamental nursery production. The objective of this study was to evaluate Brassicaceae cover crops for susceptibility to most destructive soil-borne pathogens of nursery production, P. nicotianae and R. solani, to identify effective cover crops that can be used in the biofumigation process in woody ornamental nursery production. Brassica species intended to be used in the fresh market or biofimigation were screened for their susceptibility to R. solani and P. nicotiane in an environmentally controlled greenhouse. At the end of experiments, plant growth data (plant height, width and fresh weight), total damping-off were recorded, and cover crop root systems were assessed for disease severity using a scale of 0–100% roots affected. Among the tested 15 cover crops in the Brassicaceae family, oilseed radish (Raphanus sativus L.), yellow mustard “White Gold” (Sinapis alba L.), turnip “Purple Top Forage” (Brassica rapa L.), arugula “Astro” (Eruca vesicaria (L.) Cav. ssp. sativa (Mill.) Thell.), mighty mustard^® “Pacific Gold” (B. juncea (L.) Czern.), brown mustard “Kodiak” (B. juncea (L.) Czern.), rape “Dwarf Essex” (B. napus L.) and mustard green “Amara” (B. carinata A. Braun) showed numerically lower root rot disease severity and total damping-off in topsoil which had pre-existing populations of R. solani or P. nicotinanae compared to other cover crops. Since these above mentioned Brassicaceae crops shows the ability to withstand the higher disease pressure from R. solani and P. nicotinanae under the greenhouse conditions they can be used in the further experiments to evaluate their ability in biofumigation. Further research is necessary to evaluate the performance of these cover crops under the field conditions.     

哈茨木霉的培养及其对烟草疫霉生长的抑制研究

哈茨木霉是一类重要的植病生防因子。哈茨木霉TH-1分别在PDA培养基、麦芽糖培养基、查氏培养基和琼脂培养基上培养均能产孢,其中PDA培养基为最适培养基。PDA培养基上,菌丝生长适宜温度27．5℃～35℃,最适温度32．5℃,产孢最适温度27．5℃。菌丝生长适宜pH值为3～7,产孢适宜pH值为5-9,生长与产孢最适pH值为5。光照对菌丝生长影响不大但明显影响菌株的产孢数量,光照时间越长产孢量越大。对峙培养试验表明TH-1明显抑制疫霉菌的生长速率,其无菌滤液明显抑制烟草疫霉菌游动孢子的萌发,并抑制游动孢子芽管的伸长,TH-1对游动孢子萌发的相对抑制率为12．7％,对芽管生长长度的相对抑制率为63．1％。水解酶平板活性测定显示,TH-1产生β-1,3葡聚糖酶与纤维素酶,从而使烟草疫霉菌细胞壁的消解,产生非挥发性抗生素抑制烟草疫霉菌孢子萌发,但对菌丝生长影响不大。     

Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

Two extracellular serine proteinases with molecular masses of about 53–55 and 70–72 kDa, were purified from Arthrobacter nicotianae 9458 and characterized. The enzymes differed with respect to temperature optimum, 55–60 and 37°C, respectively, tolerance to low values of pH and temperature, heat stability, sensitivity to EDTA and sulfhydryl blocking agents, and hydrophobicity. Both proteinases were optimally active in the pH range of 9.0 and 9.5, had considerable activity at pH 6.0 on α_s1- and β-caseins, and tolerated NaCl over 5%. Specificity on casein fractions was generally similar and β-casein was more susceptible to hydrolysis than α_s1-casein. The proteinases of Arthrobacter spp. may play a significant role in ripening of the smear surface-ripened cheeses.     

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.     

A simple in-vitro 'wet-plate' method for mass production of Phytophthora nicotianae zoospores and factors influencing zoospore production

A simple in-vitro ‘wet-plate’ method for mass-producing Phytophthora nicotianae zoospores at ≥ 1.0 × 10⁶ zoospores/ml is described. Temperature critically affected zoospore production; 22 °C was optimum, while 36 °C was completely inhibitory. Zoospores being the most important propagule of P. nicotianae, temperature of recycled irrigation water may be manipulated to reduce diseases in irrigated nursery crops.