Characterization of the programmed cell death induced by metabolic products of Alternaria alternata in tobacco BY-2 cells

Alternaria alternata has received considerable attention in current literature and most of the studies are focused on its pathogenic effects on plant chloroplasts, but little is known about the characteristics of programmed cell death (PCD) induced by metabolic products (MP) of A. alternata, the effects of the MP on mitochondrial respiration and its relation to PCD. The purpose of this study was to explore the mechanism of MP-induced PCD in non-green tobacco BY-2 cells and to explore the role of mitochondrial inhibitory processes in the PCD of tobacco BY-2 cells. MP treatment led to significant cell death that was proven to be PCD by the concurrent cytoplasm shrinkage, chromatin condensation and DNA laddering observed in the cells. Moreover, MP treatment resulted in the overproduction of reactive oxygen species (ROS), rapid ATP depletion and a respiratory decline in the tobacco BY-2 cells. It was concluded that the direct inhibition of the mitochondrial electron transport chain (ETC), alternative pathway (AOX) capacity and catalase (CAT) activity by the MP might be the main contributors to the MP-induced ROS burst observed in tobacco BY-2 cells. The addition of adenosine together with the MP significantly inhibited ATP depletion without preventing PCD; however, when the cells were treated with the MP plus CAT, ROS overproduction was blocked and PCD did not occur. The data presented here demonstrate that the ROS burst played an important role in MP-induced PCD in the tobacco BY-2 cells.     

Volatilization of Selenium by Alternaria alternata

Seleniferous water continues to be a serious problem to wildlife in the central valley of California. Water samples collected from Kesterson Reservoir, Peck Ranch, and Lost Hills evaporation pond facilities contained between 0.005 and 5 mg of Se per liter. The objective of this study was to isolate Se-methylating organisms in evaporation pond water and to assess, through enrichment and manipulation of their optimal growth parameters, the environmental factors which govern microbial Se methylation. Alternaria alternata was isolated as an active Se-methylating organism. The volatile product was identified as dimethylselenide. The effects of pH, temperature, Se substrates, and methyl donors on the ability of A. alternata to methylate Se were investigated in liquid medium containing 100 mg of Se per liter. The optimum pH and temperature for methylation were 6.5 and 30 degrees C, respectively. Selenate and selenite were methylated more rapidly than selenium sulfide and various organic Se compounds (6-selenoguanosine, 6-selenoinosine, seleno-dl-methionine, and 6-selenopurine). l-Methionine and methyl cobalamine (0.1 muM) stimulated dimethylselenide production. This study demonstrates that Se-methylating organisms are present in evaporation pond water and are capable of liberating substantial quantities of Se in the volatile dimethylselenide form. By determining the optimum environmental conditions which stimulate volatilization, it may be possible to design a way to remove Se from seleniferous water in situ.     

Ammonia-related metabolism of tobacco varieties differing in their susceptibility to Alternaria alternata

Some studies report that ammonia is an important factor of disease development in tobacco plants and various post-harvest fruits. Four tobacco (Nicotiana tabacum L.) varieties resistant or susceptible to Alternaria alternata (Fries) Keissler, a tobacco pathogenic fungus, were used to investigate whether there are differences in ammonia accumulation and the related metabolism of senescing leaves. The results showed that: (a) the leaves of susceptible varieties had significantly higher apoplastic [NH ₄ ⁺ ], pH, and ammonia emission potential (??-values) than resistant varieties during the period from 40 to 60 days of leaf age; (b) leaf tissue [NH ₄ ⁺ ] and total N concentrations in the tobacco varieties were not in line with their susceptibility or resistance to disease; (c) the increases in the apoplastic pH, ??-values, and leaf [NH ₄ ⁺ ] occurred in parallel with a significant decline in glutamine synthetase activity. Compared with the resistant varieties, apoplastic pH values and ?? values were increased more rapidly in the susceptible varieties due to a steeper decline in glutamine synthetase activity and a slower increase in glutamate dehydrogenase activity. In conclusion, NH₃ accumulation or NH3-dependent alkalinization rather than [NH ₄ ⁺ ] and total N appears to be mainly attributed to the enhanced susceptibility of tobacco plants to A. alternata.     

不同化合物对烟草赤星病菌毒素的钝化研究

为确定几种不同化合物对烟草赤星病菌产生的毒素毒性的影响,作者采用摩擦接种处理法、浇根处理法、种子根处理法3种生物测定方法,确定了以烟草种子根处理法作为烟草赤星病菌的毒性测定方法。用6种化合物分别以5种不同浓度与AT毒素混合接种,根长测定结果表明：氯化铁、硫酸亚铁、硫酸锰和柠檬酸在0．02—20mg/mL浓度范围内、钼酸钠在0．02—2mg/mL浓度范围内对对烟草赤星病菌毒素有明显钝化作用;所有处理中钼酸钠0．02mg/mL、柠檬酸0．02mg/mL浓度处理效果最好,其次为钼酸钠0．2mg／mL处理浓度。     

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen inhibition of mycotoxin production by Alternaria alternata

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

Biotransformation of arenobufagin and cinobufotalin by Alternaria alternata

Abstract

The biotransformation of arenobufagin (1) and cinobufotalin (2), isolated from the natural medicine Chan Su, by Alternaria alternata AS 3.4578 was carried out. Incubation of 1 and 2 afforded six metabolites: 3-oxo-arenobufagin (1a), ψ-bufarenogin (1b), 3-oxo-ψ-bufarenogin (1c), 3-oxo-Δ⁴-derivative of cinobufotalin (2a), 3-oxo-cinobufotalin (2b) and 12β-hydroxycinobufotalin (2c). Among them, metabolites 1a, 1c and 2c are new compounds and their structures were characterized on the basis of their spectroscopic data (NMR, MS and IR). Compounds 1, 2, 1b, 2a and 2b were evaluated for their cytotoxicity against HepG2 and MCF-7 human cancer cells, and all of them showed significant inhibitory activities.     

Initial stages in the onset of senescence in tobacco leaves

A marked loss of leucine ¹⁴C incorporation occurred in chloroplasts isolated from Nicotiana rustica L. leaves exposed to 24 hours of darkness. This loss is not due to an initial decline in RNA-synthesis potential of the chloroplasts, as was inferred from the extent of UTP incorporation by the isolated chloroplasts. Upon reillumination of the leaves, leucine incorporation by the isolated chloroplasts reverted to its original level within 3 to 4 hours, hence it is doubtful whether the period of 24 hours after detachment should be regarded as the initial phase of leaf senescence.     

Nitrogen inhibition of mycotoxin production by Alternaria alternata.

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

链格孢Alternaria alternata侵染导致瓜类幼苗白化的机制

用分离自无症状瓜类植物的27株链格孢属Alternaria真菌接种黄瓜,有14株引起了黄瓜幼苗的白化,这些菌株均属于链格孢A. alternata。为揭示A. alternata引起黄瓜幼苗白化的机制,对代表菌株5F29经固体发酵后提取其次级代谢产物,将粗提物按一定量混入基质进行盆栽实验。结果表明,黄瓜、丝瓜和甜瓜对该粗提物敏感,出现白化苗;小白菜和辣椒对粗提物不敏感,生长正常。植物幼苗用粗提物处理的结果与用产生菌接种的结果相一致。粗提物经硅胶柱层析、葡聚糖凝胶柱层析及高效液相色谱（HPLC）等方法分离纯化得到一种活性化合物,经测定该化合物引起黄瓜幼苗白化的最低浓度为12.5μmol/L。该化合物在此浓度下可引起黄瓜幼苗生长点白化,在15μmol/L的浓度下可引起黄瓜幼苗大部分组织白化,致使幼苗很快死亡。应用核磁共振和质谱技术,确定了该化合物的结构,鉴定为一种植物毒素——腾毒素（tentoxin）。研究结果揭示了A. alternata引起瓜类幼苗白化的机制以及不同植物对于腾毒素的不同敏感性。     

不同种植模式对烤烟根系和叶片衰老特性的影响

以烤烟品种K326为试材,采用大田试验研究了广东南雄地区不同种植模式（单行种植+前期垄体覆盖地膜、单行种植+前期垄体覆盖地膜+后期垄体覆盖稻草、双行凹垄种植+前期垄体覆盖地膜和双行凹垄种植+前期垄体覆盖地膜+后期垄体覆盖稻草）对烤烟根系和叶片衰老特性,以及烤后烟叶经济性状的影响.结果表明：与其他种植模式相比,双行凹垄种植+前期垄体覆盖地膜+后期垄体覆盖稻草处理显著促进了各时期烤烟根系生长,根系活力提高;叶片叶绿素含量和保护酶(SOD、POD)活性均较高,而MDA含量较低,烤后烟叶的产量、产值、均价和上等烟比例等经济性状均显著高于其他处理.双行凹垄种植+前期垄体覆盖地膜+后期垄体覆盖稻草处理可以改善烤烟根系和叶片衰老特性,提高烤后烟叶经济性状,是广东南雄地区优质烟叶生产的有效种植模式.     

L型氨基酸对烟草赤星病菌毒素毒性的影响

为确定不同氨基酸对烟草赤星病菌产生的毒素毒性的影响,作者采用摩擦接种处理法、浇根处理法、种子根处理法三种生物测定方法,确定了以烟草种子根处理法作为烟草赤星病菌的毒性测定方法。用12种氨基酸分别以5种不同浓度与AT毒素混合接种,根长测定结果表明：L-谷氨酰胺,L-天冬酰胺,L-组氨酸所有供试浓度均增强病原菌毒素的毒性,而其余9种L型氨基酸对烟草赤星病菌GL-6产生的毒素有抑制作用,其中L-甲硫氨酸、L-亮氨酸、L-赖氨酸适宜浓度可明显降低供试病原菌毒素对烟草的致病性,与对照差异极显著,其有效浓度分别为0．02mg／ml、0．02mg／ml、0．02～2mg／ml。     

Production of a Thermostable beta-d-Galactosidase by Alternaria alternata Grown in Whey

In the course of exploring new microbial sources of extracellular beta-d-galactosidase (EC. 3.2.1.23), Alternaria alternata was found to excrete elevated quantities of a thermostable form of the enzyme when cultivated in whey growth medium. Optimum cultural conditions for maximum enzyme production were a whey lactose concentration of 6%, supplementation of the medium with 0.050 M (NH(4))(2)SO(4), an inoculum size of 10 conidia per ml, and a cultivation time at 28 to 30 degrees C of 5 days. The fungus utilized whey lactose for the production of the enzyme most efficiently, and the observed maximum yield, 280 nanokatals of hydrolyzed o-nitrophenyl-beta-d-galactopyranoside per g of whey lactose, was comparable to maximum yields reported for certain commercial fungi. The optimum pH and temperature of the enzymatic reaction were 4.5 to 5.5 and 60 to 70 degrees C, respectively, and the enzyme lost half of its activity when heated at 65 degrees C for 84 min. These properties make the enzyme particularly suitable for processing acid and less-acid (pH 5 to 6) dairy products and by-products.     

Control by imazalil of fruit storage rots caused by Alternaria alternata

The fungicide imazalil has potential value for controlling post-harvest diseases caused by Alternaria alternata in pears, apples and persimmons. Imazalil was active in vitro on germination and hyphal growth (ED₅₀) at 1.4 and 0.5 μg/ml, respectively. Protectant action of the fungicide lasted only 1 day after treatment. At 1000 μg/ml the fungicide inhibited development of Alternaria rot in inoculated apples at 0 °C and in naturally infected pears and persimmons at–1 °C.     

Simultaneous production of glucose oxidase and catalase by Alternaria alternata

Summary A number of factors affecting simultaneous production of cell-bound glucose oxidase and catalase by the fungus Alternaria alternata have been investigated. Consecutive optimization of the type and concentration of nitrogen and carbon source, the initial pH and growth temperature resulted in a simultaneous increase in glucose oxidase and catalase by 780% and 68% respectively. Two second-order equations, describing the combined effect of pH and temperature on the activity of each enzyme, revealed that glucose oxidase had its optima at pH 7.9 and 32.3°C and catalase at pH 8.5 and 18.1°C. Under certain growth conditions, yields as high as 23.5 and 18,100 units/g carbon source for glucose oxidase and catalase, respectively, were simultaneously obtained.Offprint requests to: B. J. Macris     

Postharvest biocontrol of Alternaria alternata in Chinese winter jujube by Rhodosporidium paludigenum

Aims:  This study was conducted to measure the efficacy of the marine antagonist Rhodosporidium paludigenum in the suppression of postharvest decay of Chinese winter jujube caused by Alternaria alternata and to explore the possible mode of action involved.
Methods and Results:  The efficacy of controlling postharvest diseases by R. paludigenum was examined. Rapid yeast colonization of wounds was observed during the first 48 h at 25°C. The yeast at 1 × 10⁸ cells ml⁻¹ of washed cells suspension provided better control of A. alternata than any other treatment. The concentration of the antagonist had significant effects on biocontrol effectiveness: as the concentration of R. paludigenum was increased, the disease incidence decreased. Meanwhile, R. paludigenum significantly inhibited the natural development of decay and did not damage fruit quality parameters including lightness values, hue angle, firmness, soluble solids, ascorbic acid and titratable acidity in 21 days' storage at 25°C.
Conclusions:  Rhodosporidium paludigenum was effective in controlling postharvest decay of Chinese winter jujube and did not impair fruit quality parameters.
Significance and Impact of the Study:  Rhodosporidium paludigenum can be used as a nonchemical agent in postharvest biological control of Chinese winter jujube.     

Novel beta-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.     

Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by ABA. Senescence of rice leaves was determined by the decrease of protein content. ABA treatment resulted in (1) induction of leaf senescence, (2) increase in H2O2 and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase). All these ABA effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-alpha-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA + NaNO2] were effective in reducing ABA-induced leaf senescence. PBN prevented ABA-induced increase in the contents of H2O2 and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on ABA-promoted senescence, ABA-increased H2O2 content and lipid peroxidation, ABA-decreased GSH and AsA, and ABA-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of ABA-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H2O2.