Yeasts as biological agents to control Botrytis cinerea

Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains. A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B. cinerea strains. Pichia membranifaciens, P. anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains. In small-scale trials, post-harvest application of P. membranifaciens CYC 1106 to apple wounds inhibited B. cinerea CYC 20010. Purified killer toxin from P. membranifaciens CYC 1106 inhibited B. cinerea CYC 20010. Results indicated that certain yeasts, or their toxins such us P. membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B. cinerea.     

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.     

土壤养分对菊科一年生入侵种和本地种繁殖性状的影响

入侵植物繁殖性状的研究可为揭示植物入侵机制提供重要的科学依据。为研究土壤养分对入侵植物和本地植物繁殖性状的影响, 并进一步研究养分添加是否更能促进入侵植物的繁殖能力, 我们设置了低、高两个养分水平, 通过同质园实验比较了不同土壤养分对假臭草(Praxelis clematidea)、胜红蓟(Ageratum conyzoides)、三叶鬼针草(Bidens pilosa) 3种菊科一年生入侵种和夜香牛(Vernonia cinerea)、一点红(Emilia sonchifolia)、墨旱莲(Eclipta prostrata) 3种本地种繁殖性状的影响。研究结果显示, 养分添加提高了6种菊科植物的开花株高、株高、地上生物量、单粒种子重量、总花序数、每花序种子数、总种子数量、总种子重量, 并使开花时间提前、花期延长。养分添加对入侵种的开花株高和单粒种子重量的提高幅度要比本地种更显著。相对于部分本地植物(夜香牛、墨旱莲), 养分添加更能促进部分入侵植物(假臭草、胜红蓟)的繁殖能力。三叶鬼针草和一点红的总种子数量和总种子重量在两种土壤养分水平下均较小。本地种墨旱莲的总种子数量和总种子重量在低养分条件下高于3个入侵种。这些结果表明, 高土壤养分仅能促进部分入侵植物相对于部分本地植物的繁殖能力。     

芳樟醇对灰葡萄孢生长的影响及对番茄灰霉病的防控效果

通过平板抑菌试验和孢子萌发试验研究了芳樟醇对灰葡萄孢的生长抑制作用,并通过盆栽试验进一步验证了芳樟醇对番茄灰霉病的防控效果。结果表明: 芳樟醇能够显著抑制灰葡萄孢菌丝的生长,半最大效应浓度(EC₅₀)值为0.581 mL·L^-1。孢子萌发试验中,芳樟醇能够有效抑制灰葡萄孢孢子的萌发,并表现出浓度依赖性。芳樟醇处理提高了灰葡萄孢菌菌丝体的相对电导率和丙二醛(MDA)含量,说明芳樟醇可引起氧化损伤效应导致灰葡萄孢菌的膜系统被破坏;芳樟醇处理后灰葡萄孢菌中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性较对照组分别下降了27.4%、68.9%和26.0%,说明芳樟醇抑制了灰葡萄孢菌体内的抗氧化系统。盆栽试验结果显示,芳樟醇处理的病斑直径较对照组显著降低;番茄叶片中的SOD、CAT、POD、多酚氧化酶和苯丙氨酸解氨酶活性均显著高于对照组;而MDA含量下降了41.5%,说明芳樟醇可减轻灰葡萄孢菌对番茄植株造成的氧化损伤以提高植物抗病性。综上,芳樟醇对灰葡萄孢的生长有显著抑制作用并对番茄灰霉病有较好的防治效果,研究结果可为开发新型植物源抑菌剂防控番茄灰霉病提供理论依据。     

Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Tomato responds to green peach aphid infestation with the activation of trehalose metabolism and starch accumulation

The disaccharide trehalose and trehalose-6-phosphate that are present in trace amounts are suggested to have a signaling function in plants. Recently, it was demonstrated that trehalose metabolism contributes to Arabidopsis thaliana defense against the green peach aphid (GPA; Myzus persicae Sülzer), an important insect pest of a large variety of plants. TPS11 (TREHALOSE PHOSPHATE SYNTHASE11)-dependent trehalose metabolism was shown to curtail GPA infestation by promoting starch accumulation and expression of the PAD4 (PHYTOALEXIN-DEFICIENT4) gene, which has important roles in regulating antibiosis and antixenosis against GPA. Here we show that trehalose metabolism is similarly activated in leaves of GPA-infested tomato (Solanum lycopersicum) plants and likely contributes to tomato defense against GPA. GPA-infested leaves of tomato accumulated trehalose, which was accompanied by the transient upregulation of SlTPS11, a homolog of the Arabidopsis TPS11. GPA-infestation was also accompanied by starch accumulation and the upregulation of SlPAD4, the tomato homolog of Arabidopsis PAD4. Furthermore, trehalose application induced SlPAD4 expression and starch accumulation, and curtailed GPA infestation, suggesting that like in Arabidopsis trehalose contributes to tomato defense against GPA.     

西藏马鹿与同域野生有蹄类、家畜草青期食性比较

西藏马鹿为我国特有种,曾一度被认定为野外灭绝。在其生境中有多种食性相近的野生有蹄类及家畜同域分布,探究青藏高原特殊环境下西藏马鹿与这些有蹄类的食物资源种间竞争与共存关系十分必要。2013年和2014年8—9月,采用粪便显微分析法确定了桑日县西藏马鹿自然保护区西藏马鹿、白唇鹿、藏原羚、牦牛、山羊草青期食物组成,并比较分析了西藏马鹿与同域野生有蹄类、家畜之间的食物重叠和分化状况。结果表明: 西藏马鹿与其他有蹄类具有相似的可食植物种类组成,但所占食谱比例不同。西藏马鹿主要可食植物多为其他有蹄类次要可食植物,其中弱小火绒草为西藏马鹿(在动物食谱中的百分比为11.2%)与牦牛(10.2%)共同的主要食物,西藏柳为西藏马鹿(9.6%)和白唇鹿(11.4%)共同的主要食物。从科级水平来看,豆科植物为西藏马鹿(21.4%)与藏原羚(42.5%)的共同主要可食植物,莎草科植物为白唇鹿(49.2%)、牦牛(33.4%)、山羊(50.3%)的共同主要可食植物。菊科植物作为西藏马鹿(29.6%)的主要可食植物,也是白唇鹿(7.6%)、藏原羚(11.6%)、牦牛(17.3%)和山羊(14.1%)的次要可食植物。禾本科植物作为西藏马鹿(7.1%)次要可食植物,较其他4种有蹄类(白唇鹿13.6%、藏原羚12.3%、牦牛11.5%、山羊16.0%)所占比例低。西藏马鹿与其他有蹄类食物重叠度均高于0.5,与牦牛的重叠度最高(0.65)。西藏马鹿食物多样性指数(1.32)、均匀度指数(0.37)和生态位宽度指数(15.79)均较高。与2007—2008年相比,西藏马鹿食物组成变化较大,豆科植物比例增大,莎草科植物比例降低,食物质量总体有所提高。西藏马鹿与家畜之间存在较高的食物资源竞争,这将进一步影响西藏马鹿的分布范围和生存空间。     

Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense

Mulberry leaves (Morus spp.) exude latex rich in sugar-mimic alkaloids, 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ), as a defense against herbivorous insects. Sugar-mimic alkaloids are inhibitors of sugar-metabolizing enzymes, and are toxic to the Eri silkworm, Samia ricini, a generalist herbivore, but not at all to the domesticated silkworm, Bombyx mori, a mulberry specialist. To address the phenomena, we fed both larvae diets containing different sugar sources (sucrose, glucose or none) with or without sugar-mimic alkaloids from mulberry latex. In S. ricini, addition of sugar-mimic alkaloids to the sucrose (the major sugar in mulberry leaves) diet reduced both growth and the absorption ratio of sugar, but it reduced neither in B. mori. The midgut soluble sucrase activity of S. ricini was low and inhibited by very low concentrations of sugar-mimic alkaloids (IC₅₀=0.9–8.2 μM), but that of B. mori was high and not inhibited even by very high concentrations (IC₅₀>1000 μM) of sugar-mimic alkaloids. In S. ricini, the addition of sugar-mimic alkaloids to the glucose diet still had considerable negative effects on growth, although it did not reduce the absorption ratio of glucose. The hemolymph of S. ricini fed sugar-mimic alkaloids contained sugar-mimic alkaloids. The trehalose concentration in the hemolymph increased significantly in S. ricini fed sugar-mimic alkaloids, but not in B. mori. The trehalase activities of S. ricini were lower and inhibited by lower concentrations of sugar-mimic alkaloids than those of B. mori. These results suggest that sugar-mimic alkaloids in mulberry latex exert toxicity to S. ricini larvae first by inhibiting midgut sucrase and digestion of sucrose, and secondly, after being absorbed into hemolymph, by inhibiting trehalase and utilization of trehalose, the major blood sugar. Further, our results reveal that B. mori larvae evolved enzymatic adaptation to mulberry defense by developing sucrase and trehalase that are insensitive to sugar-mimic alkaloids.     

典型黑土区主要树种根系构型特征及其对固土能力的影响

为量化典型黑土区主要树种根系构型特征,探究其对固土能力的影响,以该区分布较广的榆叶梅、小叶锦鸡儿、白桦、糖槭、红皮云杉、樟子松单株个体为研究对象,采用全根挖掘和WinRHIZO Pro LA2004分析系统相结合对其根系空间分布、几何形态、分形等特征进行测定,同时采用原位整株根系拉拔的方法量化根系垂直拉拔力。结果表明: 榆叶梅以倾斜根为主,小叶锦鸡儿、白桦、糖槭和红皮云杉以水平根为主,樟子松根系在水平和垂直分布上较为均衡;除白桦总根表面积和红皮云杉总根长外,灌木树种总根长、总根表面积显著大于乔木,落叶阔叶乔木总根长、总根表面积显著大于针叶常绿乔木,白桦总根体积显著大于小叶锦鸡儿、糖槭、红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和白桦根系分形维数和分形丰度显著大于红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和糖槭整株根系平均最大垂直拉拔力显著大于白桦、樟子松和红皮云杉。主要受根系总根长、总根表面积和倾斜根数量的影响,榆叶梅、小叶锦鸡儿和糖槭根系表现出较强的固土能力,可作为典型黑土区水土保持植被构建中优先选择的树种。     

Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Co-regulation of indole glucosinolates and camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6 signaling pathways

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.     

Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae

Gamma‐aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen‐responsive mitogen‐activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst‐avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst‐avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst‐avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst‐avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

PROTEIN S-ACYL TRANSFERASE 13/16 modulate disease resistance by S-acylation of the nucleotide binding,leucine-rich repeat protein R5L1 in Arabidopsis

Nucleotide binding, leucine-rich repeat(NB-LRR)proteins are critical for disease resistance in plants,while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000(Pst DC3000). Of the five mutants that were hyper-...     

Enantiomeric oxidation of organic sulfides by the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride

The biotransformations of a series of substituted sulfides were carried out with the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride. Several products underwent microbial oxidation of sulfide to sulfoxide with medium to high enantiomeric purity. With regard to sulfoxide enantioselectivity, the (R)-enantiomer was favoured in biotransformations by T. viride and E. lata while the (S)-enantiomer was favoured in those by B. cinerea. A minor amount of sulfone product was also obtained.     

In vitro effects of crude extracts of Parkia biglobosa (Mimosaceae), Stereospermum kunthianum (Bignoniaceae) and Biophytum petersianum (Oxalidaceae) on corticosteroid secretion in rat

Previous studies conducted in guinea pig, rat and rabbit have revealed that crude extracts from Parkia biglobosa, Stereospermum kunthianum and Biophytum petersianum exert hypotensive and/or hypoglycemic activities. Since corticosteroids are involved in the control of arterial blood pressure and glycemia, we have investigated the possible effects of these plant extracts on rat adrenal tissue in vitro. Short-term administration of crude semi-ethanolic extracts of P. biglobosa and S. kunthianum to perifused rat adrenal tissue did not induce any significant changes in corticosteroid output. Conversely, the B. petersianum extract caused a dose-dependent increase in corticosterone and aldosterone secretion. Repeated infusions or prolonged administration of B. petersianum extract did not produce any apparent attenuation of the steroid response. Altogether, these data indicate that a semi-ethanolic extract of B. petersianum dose-dependently stimulates corticosterone and aldosterone secretion in rat without any desensitization phenomenon.     

一株谦逊迷孔菌Daedalea modesta的分离及其杀线虫的作用

为了研究野生担子菌对线虫的作用效果,从腐木桩上采集担子菌,应用常规组织分离法获得纯培养菌丝,以全齿复活线虫作靶标,初步筛选到1株对线虫有高活性的菌株,应用形态学方法和分子生物学方法进行了种的鉴定,测定了其对全齿复活线虫Panagrellus redivivus、松材线虫Bursaphelenchus xylophilus、爪哇根结线虫Meloidogyne javanica的杀线虫活性,并测定了其在不同条件下的发酵液对全齿复活线虫、松材线虫和爪哇根结线虫二龄幼虫的作用效果。经鉴定该菌株为谦逊迷孔菌Daedalea modesta,其菌丝在平板上能够迅速杀死线虫,平板菌落上接种线虫48h,菌丝对全齿复活线虫、松材线虫和爪哇根结线虫的致死率分别为95.61%、85.75%和100%;其马铃薯葡萄糖液体发酵液25℃处理全齿复活线虫、松材线虫和爪哇根结线虫二龄虫24h线虫死亡率均达100%,发酵液稀释10×时,对爪哇根结线虫仍有致死效果。本研究为该菌株用于线虫防治的进一步研究提供了科学参考。