酵母拮抗菌的抑病机理及生防效力改良的研究

相对于酵母拮抗菌的使用来说，人们对其作用机理了解得还不是很清楚。而了解拮抗菌的抑菌机理却是增强拮抗菌的生防效果以及进行拮抗菌筛选标准的重要前提。本文主要研究了酵母拮抗菌Pichia membranefaciens、Cryptococcus albidus以及Crytococcus laurentii对水果采后软腐病、褐腐病以及青霉病的防治效果，拮抗菌与病原菌之间的相互作用，并对酵母拮抗菌与外源物质配合使用，以及通过遗传改良途径来提高酵母拮抗菌生防能力等进行了初步研究。实验结果如下： 1、酵母拮抗菌P. membranefaciens、C. albidus以及C. laurentii能在果实伤口大量繁殖。采用扫描电镜技术，观察发现在桃果实伤口处P. membranefaciens能紧密地吸附在软腐病菌Rhizopous stolonfier的菌丝体上;C. laurentii与青霉病菌Penicillium expansum在苹果果实伤口处也存在着直接的拮抗作用;但P. membranefaciens和C. albidus对P. expansum的直接作用不明显。 2、酵母拮抗菌P. membranefaciens能够有效地抑制甜樱桃果实在常温和低温贮藏条件下褐腐病的发生。在常温贮藏条件下，P. membranefaciens和褐腐病菌Monilinia fracticola 处理都能够提高果实β-1,3-葡聚糖酶、POD、以及PAL酶的活性，但在低温贮藏条件下，拮抗菌和病原菌处理对甜樱桃果实β-1,3-葡聚糖酶、POD酶活性的升高有促进作用，对PAL和PPO酶活性的诱导作用不明显。 3、梨果实采后经过水杨酸，CaCl2，UV辐射和草酸等各种激发子处理以后，再接种病原菌Alternaria alternata，可以显著降低梨果实的发病率。其中，水杨酸处理的果实发病率最低。不同的激发子均可以诱导梨果实β-1,3-葡聚糖酶、POD、PAL和PPO酶活性的升高，但对果实乙烯含量的影响不明显。 4、氨基糖甙类抗菌素G418能够抑制P. membranefaciens的生长，其最低抑制浓度为100g ml-1。将G418抗性基因Neor插入到酵母-大肠杆菌穿梭表达载体pFL61中，构建PGK启动子驱动的表达载体pFL61-neo，利用醋酸锂转化法转化P. membranefaciens。酵母转化子在非选择性培养条件下连续生长50代后，仍有67.87%的细胞保留该质粒。这表明穿梭表达载体pFL61-neo能稳定地存在于P. membranefaciens中，并且该酵母细胞能有效地识别PGK启动子和终止子指导Neor的表达。 5、酵母拮抗菌C. laurentii和Rhodotorula glutinis与2%的碳酸氢钠混合使用，对冬枣果实青霉病的防治效果明显比单独使用拮抗菌或化学物质的防病效果好。其中，107CFU ml-1的拮抗菌与238 mmol l-1的碳酸氢钠配合使用可以达到单独使用108CFU ml-1拮抗菌的防病效果。另外，钼酸铵作为一种添加剂也能提高R. glutinis对梨果实青霉病和黑霉病的防治效果，但将钼酸铵与Trichosporon sp.配合使用的防病效果不明显。碳酸氢钠和钼酸铵在果实伤口对酵母拮抗菌的生长都有一定的抑制作用。 6、酵母拮抗菌P. membranefaciens在不同碳源、氮源中生长情况表明：在几种氮源中，大豆蛋白胨、酵母提取物、牛肉浸膏对P. membranefaciens的生长有显著的促进作用，其中，大豆蛋白胨的效果最好。在检测以葡萄糖、果糖和麦芽糖作为碳源的生长实验中，发现这几种碳源都能够被拮抗菌很好的利用，其中葡萄糖的利用率最好。小球藻生长因子（CGF）能够明显地促进了P. membranefaciens的生长。但是，CGF的浓度从0.5%增加到1%并没有促进酵母菌细胞数量的增加。

The effect of carrier substrate, dose rate and time of application on biocontrol efficacy of fungal antagonists against Armillaria root rot of strawberry plants.

In a glasshouse experiment using potted strawberry plants (cv. Cambridge Favourite) as hosts, the effect of selected fungal antagonists grown on 25 or 50 g of mushroom compost containing autoclaved mycelia of Agaricus bisporus, or wheat bran was evaluated against Armillaria mellea. Another glasshouse experiment tested the effect of application time of the antagonists in relation to inoculations with the pathogen. A significant interaction was found between the antagonists, substrates and dose rates. All the plants treated with Chaetomium olivaceum isolate Co on 50 g wheat bran survived until the end of the experiment which lasted 482 days, while none of them survived when this antagonist was added to the roots of the plants on 25 g wheat bran or 25 or 50 g mushroom compost. Dactylium dendroides isolate SP had a similar effect, although with a lower host survival rate of 33.3%. Trichoderma hamatum isolate Tham 1 and T. harzianum isolate Th23 protected 33.3% of the plants when added on 50 g and none when added on 25 g of either substrate, while 66.7% of the plants treated with T. harzianum isolate Th2 on 25 g, or T viride isolate TO on 50 g wheat bran, survived. Application of the antagonists on mushroom compost initially resulted in development of more leaves and healthier plants, but this effect was not sustained. Eventually, plants treated with the antagonists on wheat bran had significantly more leaves and higher health scores. The plants treated with isolate Th2 and inoculated with Armillaria at the same time had a survival rate of 66.7% for the duration of the experiment (475 days), while none of them survived that long when the antagonist and pathogen were applied with an interval of 85 days in either sequence. C. olivaceum isolate Co showed a protective effect only, as 66.7% of the plants survived when they were treated with the antagonist 85 days before inoculation with the pathogen, while none of them survived when the antagonist and pathogen were applied together or the infection preceded protection.

Cultural techniques for improvement in biocontrol potential of fungal antagonists for biological control of Armillaria root rot of strawberry plants under glasshouse conditions

Several in vitro and in vivo experiments were conducted to develop an effective technique for culturing potential fungal antagonists (isolates of Trichoderma harzianum, Dactylium dendroides, Chaetomium olivaceum and one unidentified fungus) selected for activity against Armillaria mellea. The antagonists were inoculated onto (1) live spawn of the oyster mu shroom (Pleurotus ostreatus), (2) extra-moistened or sucrose-enriched mushroom composts containing living or autoclaved mycelia of P. ostreatus or Agaricus bisporus (button mushroom), (3) pasteurized compost with or without A. bisporus mycelium, wheat bran, wheat germ and (4) spent mushroom composts with living mycelia of A. bisporus, P. ostreatus or Lentinus edodes (the Shiitake mushroom). In one experiment, a representative antagonist (isolate Th2 of T. harzianum) was grown together with the A. bisporus mycelium, while in another one, the antagonist was first grown on wheat germ or wheat bran and then on mushroom compost with living mycelium of A. bisporus. Some of the carrier substrates were then added to the roots of potted strawberry plants in the glasshouse to evaluate their effectiveness against the disease. The antagonists failed to grow on the spawn of P. ostreatus even after reinoculations and prolonged incubation. Providing extra moisture or sucrose enrichment also did not improve the growth of Th2 on mushroom composts in the presence of living mycelia of A. bisporus or P. ostreatus. The antagonist, however, grew rapidly and extensively on mushroom compost with autoclaved mycelia, and also on wheat germ and wheat bran. Colonization of the substrates by the antagonist was positively correlated with its effectiveness in the glasshouse studies. Whereas only 33.3% of the inoculated control plants survived in one experiment monitored for 560 days, 100% survival was achieved when Th2 was applied on wheat germ or wheat bran. Growth of the antagonist alone on pasteurized or sterilized compost (without A. bisporus mycelia) and simultaneous growth of the antagonist and mushroom on pasteurized compost did not improve survival over the inoculated controls, but growth over mushroom compost with the living mycelium resulted in 50% survival rate. C. olivaceum isolate Co was the most effective, resulting in overall survival rate of 83.3% compared with only 8.3% for the inoculated and 100% for the uninoculated (healthy) controls. This antagonist gave the highest survival rate of 100% on spent mushroom compost with L. edodes. T harzianum isolate Th23, with 75% survival rate, was the most effective on spent mushroom compost with P. ostreatus, while D. dendroides isolate SP resulted in equal survival rates of 50% on all the three mushroom composts.

Use of essential oils and biocontrol cultures for the improvement of shelf-life of fresh cut products

The demand of minimally processed fruits and vegetables has increased in the last years. However, their intrinsic characteristics may favor the growth of pathogens and spoilage microbiota. The negative effects on human health reported for some traditional chemical sanitizers have justified the search for substitutes to guarantee food safety and quality. In this work we have evaluate the potential of some essential oils and their components to improve the safety and the shelf life of Lamb’s lettuce (Valerianella locusta) and apples (Golden delicious). Moreover, the effects of selected lactic acid bacteria alone or in combination with essential oils or their components, on the shelf-life and safety as well as organoleptic properties of minimally processed products, were evaluated. Since the lack of knowledge of microbial cell targets of essential oils represent one of the most important limit to the use of these molecules at industrial level, another aim of this thesis was the study of the action mechanisms of essential oils and their components. The results obtained showed the beneficial effects of the natural antimicrobials as well as the selected lactic acid bacteria on minimally processed fruit and vegetable safety and shelf-life, without detrimental effects on the quality parameters. The beneficial effects obtained by the use of the selected biocontrol agents were further increased combining them with selected natural antimicrobials. The natural antimicrobial employed induced noticeable modifications of membrane fatty acid profiles and volatile compounds produced by microbial cells during the growth. The modification of the expression in genes involved in fatty acid biosynthesis suggesting that the cytoplasmic membrane of microbial cells is one of the major cellular target of essential oils and their components. The comprehension of microbial stress response mechanisms can contribute to the scaling up of natural antimicrobials and bio-control agents at industrial level.

Pre-release evaluation of the efficacy of the leaf-sucking bug Carvalhotingis visenda (Heteroptera: Tingidae) as a biological control agent for cat's claw creeper Macfadyena unguis-cati (Bignoniaceae)

In classical weed biological control, assessing weed response to simulated herbivory is one option to assist in the prioritization of available agents and prediction of their potential efficacy. Previously reported simulated herbivory studies suggested that a specialist herbivore in the leaf-feeding guild is desirable as an effective biological control agent for cat's claw creeper Macfadyena unguis-cati (Bignoniaceae), an environmental weed that is currently a target for biological control. In this study, we tested (i) whether the results from glasshouse-based simulated herbivory can be used to prioritise potential biological control agents by evaluating the impact of a leaf-sucking tingid bug Carvalhotingis visenda (Drake & Hambleton) (Hemiptera: Tingidae) in quarantine; and (ii) the likely effectiveness of low- and high-densities of the leaf-sucking tingid after its release in the field. The results suggest that a single generation of C. visenda has the potential to reduce leaf chlorophyll content significantly, resulting in reduced plant height and leaf biomass. However, the impact of one generation of tingid herbivory on below-ground plant components, including the roots and tuber size and biomass, were not significant. These findings are consistent with results obtained from a simulated herbivory trial, highlighting the potential role of simulated herbivory studies in agent prioritisation.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.

Improvement of strategies for the management of fire blight (Erwinia amylovora). Evaluation and optimization of physical and chemical control methods, and use of decision support systems

El foc bacterià és una malaltia que afecta a plantes de la família de la rosàcies, causada pel bacteri Erwinia amylovora. El seu rang d'hostes inclou arbres fruiters, com la perera, la pomera o el codonyer, i plantes ornamentals de gran interès comercial i econòmic. Actualment, la malaltia s'ha dispersat i es troba àmpliament distribuïda en totes les zones de clima temperat del món. A Espanya, on la malaltia no és endèmica, el foc bacterià es va detectar per primer cop al 1995 al nord del país (Euskadi) i posteriorment, han aparegut varis focus en altres localitzacions, que han estat convenientment eradicats. El control del foc bacterià, és molt poc efectiu en plantes afectades per la malaltia, de manera que es basa en mesures encaminades a evitar la dispersió del patogen, i la introducció de la malaltia en regions no endèmiques. En aquest treball, la termoteràpia ha estat avaluada com a mètode d'eradicació d'E. amylovora de material vegetal de propagació asimptomàtic. S'ha demostrat que la termoteràpia és un mètode viable d'eradicar E. amylovora de material de propagació. Gairebé totes les espècies i varietats de rosàcies mantingudes en condicions d'humitat sobrevivien 7 hores a 45 ºC i més de 3 hores a 50 ºC, mentre que més d'1 hora d'exposició a 50 ºC amb calor seca produïa danys en el material vegetal i reduïa la brotació. Tractaments de 60 min a 45 ºC o 30 min a 50 ºC van ser suficients per reduir la població epífita d'E. amylovora a nivells no detectables (5 x 102 ufc g-1 p.f.) en branques de perera. Els derivats dels fosfonats i el benzotiadiazol són efectius en el control del foc bacterià en perera i pomera, tant en condicions de laboratori, com d'hivernacle i camp. Els inductors de defensa de les plantes redueixen els nivells de malaltia fins al 40-60%. Els intervals de temps mínims per aconseguir el millor control de la malaltia van ser 5 dies pel fosetil-Al, i 7 dies per l'etefon i el benzotiadiazol, i les dosis òptimes pel fosetil-Al i el benzotiadiazol van ser 3.72 g HPO32- L-1 i 150 mg i.a. L-1, respectivament. Es millora l'eficàcia del fosetil-Al i del benzotiadiazol en el control del foc bacterià, quan es combinen amb els antibiòtics a la meitat de la dosi d'aquests últims. Tot i que l'estratègia de barrejar productes és més pràctica i fàcil de dur a terme a camp, que l'estratègia de combinar productes, el millor nivell de control de la malaltia s'aconsegueix amb l'estratègia de combinar productes. Es va analitzar a nivell histològic i ultrastructural l'efecte del benzotiadiazol i dels fosfonats en la interacció Erwinia amylovora-perera. Ni el benzotiadiazol, ni el fosetil-Al, ni l'etefon van induir canvis estructurals en els teixits de perera 7 dies després de la seva aplicació. No obstant, després de la inoculació d'E. amylovora es va observar en plantes tractades amb fosetil-Al i etefon una desorganització estructural cel·lular, mentre que en les plantes tractades amb benzotiadiazol aquestes alteracions tissulars van ser retardades. S'han avaluat dos models (Maryblyt, Cougarblight) en un camp a Espanya afectat per la malaltia, per determinar la precisió de les prediccions. Es van utilitzar dos models per elaborar el mapa de risc, el BRS-Powell combinat i el BIS95 modificat. Els resultats van mostrar dos zones amb elevat i baix risc de la malaltia. Maryblyt i Cougarblight són dos models de fàcil ús, tot i que la seva implementació en programes de maneig de la malaltia requereix que siguin avaluats i validats per un període de temps més llarg i en àrees on la malaltia hi estigui present.

Improvement of the oral praziquantel anthelmintic effect by cyclodextrin complexation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Improvement of biochemical parameters in type 1 diabetic rats after the roots aqueous extract of yacon [Smallanthus sonchifolius (Poepp.& Endl.)] treatment

The aim of this study was to evaluate the effect of yacon (Smallanthus sonchifolius) (Poepp.& Endl.) on clinical parameters under diabetic conditions. The aqueous extract of yacon tuberous roots (YRAE; 0.76gfructankg-1 body weight) was prepared at the moment of each administration. Thirty-two male rats were divided into four groups (n=8): control group (C); group that received YRAE (Y); untreated diabetic group (DM1); and diabetic group treated with YRAE (Y-DM1). The diabetes mellitus was induced by streptozotocin (60mgkg-1 body weight). The animals from Y2 and Y-DM1 received YRAE by gavage, at 7-day intervals, for 30days. The aqueous extract of yacon roots decreased (p<0.05) the water and food intake in diabetic rats (Y-DM1). YRAE treatment reduced (p<0.05) glycaemia, total cholesterol, VLDL-c, LDL-c and triacylglycerol levels in diabetic rats (YRAE). HDL, urea and creatinine levels did not differ (p>0.05) between the Y and Y-DM1 groups. YRAE normalised alanine aminotransferase (ALT) activity, when comparing DM1 and Y-DM1 rats, but had no effect on lactate dehydrogenase activity (LDH). In conclusion, YRAE was sufficient for controlling water and food consumption, hyperglycaemia and dyslipidaemia, and promote the reduction of the ALT, suggesting a hepatoprotective effect in rats with STZ-induced DM1. © 2013 Elsevier Ltd.

Early improvement of psychotic symptoms with lithium monotherapy as a predictor of later response in mania

Although lithium has been the first line agent in the treatment of bipolar disorder (BD), few studies have evaluated lithium's efficacy in mania with psychosis and its association with later response. Furthermore, given the widespread concern about antipsychotic side effects, answering a question about whether lithium alone can manage to treat both psychotic and non-psychotic mania seems a very relevant one. The present study addresses the antipsychotic efficacy of lithium monotherapy in acute mania and early improvement of psychotic symptoms as a predictor of later response of manic symptoms. Forty-six patients presenting a manic episode (32 with psychotic features and 14 subjects without psychotic features) were treated for 4 weeks with lithium monotherapy and evaluated weekly using the Young Mania Rating Scale (YMRS). Subjects with rapid cycling, substance abuse/dependence, or mixed episodes were excluded. The overall antimanic efficacy of lithium in psychosis vs. non-psychosis groups was evaluated. In addition, early improvement of psychotic symptoms and its prediction of subsequent response (>50% decrease in total YMRS scores) or remission were evaluated. Lithium showed a similar efficacy in both psychosis and non-psychosis mania. Early improvement of psychotic symptoms was associated with clinical response and remission at endpoint. (C) 2012 Elsevier Ltd. All rights reserved.