拮抗植物病原真菌的纤维素/木质素降解菌株的筛选及鉴定

【背景】秸秆还田在改善土壤肥力和丰富营养等方面有重要作用,但是也存在秸秆难以快速降解利用和病原真菌病害威胁的问题。【目的】为解决还田秸秆的降解和病原真菌病害问题,从长期秸秆还田地区采集样品,从中筛选出具有降解秸秆和抑菌功能的菌株。【方法】采用稀释分离法、苯胺蓝染色法和刚果红染色法等对秸秆高效降解菌株进行筛选,通过16S rRNA基因测序及构建系统发育树进行菌株鉴定。采用对峙培养法测定筛选到的秸秆降解菌株对玉米大斑病菌(Setostphaeria turcica)、梨黑斑病菌(Alternaria kikuchiana)、马铃薯早疫病菌(Alternaria solani)、链格孢属真菌(Alternaria alternata) ACCC38230和ACCC38231等5种供试植物病原真菌的抑制作用。以玉米大斑病菌(Setostphaeria turcica)为后期供试植物病原真菌,测定拮抗菌株代谢产物的抑菌能力;通过观察拮抗菌株粗提液对玉米大斑病菌分生孢子萌发和菌丝生长的影响,从而测定菌株对病原真菌的抑制作用。【结果】从秸秆还田土壤中分离筛选获得3株高效降解纤维素及木质素菌株并命名为JY122、ZY133和JY215。通过形态学观察和16S rRNA基因测序鉴定上述菌株全部为芽孢杆菌属(Bacillus)。通过系统发育树分析结果表明,JY122与蜡样芽孢杆菌(Bacillus cereus)相似性为99.4%,ZY133与枯草芽孢杆菌(Bacillus subtilis)相似性为100%,JY215与贝莱斯芽孢杆菌(Bacillus velezensis)相似性为99.1%。平板对峙实验结果显示,JY122、ZY133和JY215菌株对不同种属的植物病原真菌均有较强的抑制作用,抑制率高达43.74%-67.54%。此外,上述芽孢杆菌代谢产物具有抑菌活性及很强的热稳定性,经95℃处理后依然具有良好的抑菌效果。【结论】筛选获得的JY122、JY133和JY215菌株具有高效降解纤维素/木质素能力,抑制多种植物病原真菌生长,代谢产物抑菌能力强且热稳定性高。这为玉米秸秆还田提供菌株资源,也为进一步解决秸秆还田难点提供了新方法和思路。     

抑制铜绿假单胞菌和金黄色葡萄球菌生物膜形成的乳杆菌筛选及机制初探

筛选得到对慢性创面中铜绿假单胞菌（Pseudomonas aeruginosa）和金黄色葡萄球菌(Staphylococcus aureus)生物膜形成具有抑制作用的乳杆菌(Loctobacillus)。测定不同种乳杆菌对铜绿假单胞菌和金黄色葡萄球菌双菌生物膜量、生长及群体感应信号分子AI-2的影响，并采用主成分分析和菌株特性综合分析确定效果最佳的菌株，最后通过荧光定量PCR的方式探究该菌株对生物膜和群体感应相关基因表达的影响。结果表明，植物乳杆菌(Lactobacillus plantarum)、卷曲乳杆菌(Loctobacillus crispatus)、嗜酸乳杆菌(Loctobacillus acidophilus)、鼠李糖乳杆菌(Loctobacillus rhamnosus)、瑞士乳杆菌(Loctobacillus helveticus)、短乳杆菌(Loctobacilkus brevis)具有不同程度的抑菌、抑制生物膜形成的能力，且同种不同株的乳杆菌抑制生物膜形成的能力也有所差异。其中，植物乳杆菌CCFM233产生的AI-2信号分子较多，具有良好的抑菌能力，可使致病菌生物膜形成量降低，铜绿假单胞菌的LasR和rhlI基因表达水平和金黄色葡萄球菌的SarA基因表达水平显著下调。本研究旨在揭示植物乳杆菌CCFM233具有抗铜绿假单胞菌和金黄色葡萄球菌感染的潜力，为其应用于慢性创面敷料提供参考。     

产胞外多糖菌株的分离鉴定及其功能研究

微生物产生的胞外多糖(exopolysaccharides, EPS)可促进大粒径土壤团聚体形成,高产EPS的菌株在土壤改良、促进作物生长方面具有较好的应用前景。【目的】从土壤样品中筛选高产胞外多糖的细菌,研究其在土壤改良、环境适应性、广谱抗病等方面的功能,为制备土壤改良型功能菌剂提供候选菌株。【方法】采用蒽酮硫酸法测定菌株胞外多糖的产量,通过形态学观察、生理生化试验及16S rRNA基因序列测定确定其分类地位,结合土壤培养试验研究菌株对土壤团聚体形成的影响。【结果】获得3株胞外多糖产量大于500 mg/L的细菌,经鉴定A-5为地衣芽孢杆菌(Bacillus licheniformis),XJ-3为萎缩芽孢杆菌(Bacillus atrophaeus),KW3-10为耐盐芽孢杆菌(Bacillus halotolerans)。菌株A-5、XJ-3、KW3-10处理后,土壤大团聚体(>0.25 mm)含量较对照分别提高了4.07、2.14和3.16倍。3株菌株对疮痂链霉菌(Streptomyces scabies)、尖孢镰刀菌(Fusarium oxysporum)、茄链格孢菌(Alternaria solani)和立枯丝核菌(Rhizoctonia solani)等多种植物病原菌具有明显的抑制效果,可耐受pH为5-9和NaCl含量1%‒9%的盐碱环境,促进植物生长,其中KW3-10的代谢产物中IAA含量为25.58 mg/L。【结论】菌株A-5、XJ-3、KW3-10可显著促进土壤团粒结构形成,具有较好的广谱抗病性和促生长特性,可作为高效复合功能菌剂的候选菌株。     

天山大峡谷原始森林黏细菌的分离鉴定及其抗菌活性

【背景】黏细菌是一类具有多细胞群体行为特征的高等原核生物,其对植物病原真菌和细菌的捕食特性使其在植物病害防治方面具有重要的应用潜力。【目的】探究乌鲁木齐天山大峡谷原始森林可培养黏细菌的多样性并分析其抗菌活性,为发掘黏细菌生防菌株奠定基础。【方法】以天山大峡谷原始森林采集的土样和腐木为分离材料,采用兔粪诱导法和被捕食菌诱导法从中分离纯化黏细菌菌株,结合形态学观察、生理生化测定和16S rRNA基因序列分析确定其分类地位,并以6种植物病原真菌[大丽轮枝菌(Verticillium dahliae)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. vasinfectum)、拟轮枝链孢霉(Fusarium verticillioides)、立枯丝核菌(Rhizoctonia solani)、黄色镰刀菌(Fusarium culmorum)、细极链格孢菌(Alternaria tenuissima)]和1种植物病原细菌[梨火疫病菌(Erwinia amylovora)]为靶标菌,通过平板对峙法和菌苔捕食法测定其抗菌活性。【结果】从采集的样品中分离出70株菌株,经纯化后获得36株黏细菌纯培养物。经鉴定隶属于4个属,黏球菌属(Myxococcus) 30株、孢囊杆菌属(Cystobacter) 3株、珊瑚球菌属(Corallococcus) 2株和原囊菌属(Archangium) 1株。抗菌活性分析显示,本研究获得的36株黏细菌至少对2种植物病原真菌有抗菌活性,表现出广谱的抗真菌活性,初步筛选出一株菌株NSE37-1兼具广谱和高效抗真菌活性;供试的15株黏细菌对梨火疫病菌均具有捕食活性,初步筛选出一株对梨火疫病菌具有较强捕食能力的黏细菌菌株NSE25。【结论】天山大峡谷可培养黏细菌资源比较丰富,黏球菌属是该地区可培养黏细菌菌群中的优势菌。分离纯化出的黏细菌菌株均表现出广谱的抗植物病原菌活性,具有进一步研究和开发的潜在价值。     

枸杞岛海域表层水真菌群落多样性研究

通过扩增子测序,分析了枸杞岛附近海域表层水中真菌群落组成及其多样性特征。结果表明,枸杞岛周围海域真菌群落组成稳定,优势真菌菌门为子囊菌门(Ascomycota),优势菌纲为座囊菌纲(Dothideomycetes),优势真菌属包括Hortaea、青霉菌属(Penicillium)、曲霉菌属(Aspergillus)。其中丰度最高的OTU1765属于威尼克外瓶霉（Hortaea werneckii）,经FUNGuild功能预测发现这是一种潜在的人类和植物病原真菌。枸杞岛附近海域表层水中真菌群落的生态功能组成比较丰富,主要为植物病原菌和未明确的腐生真菌。研究结果揭示了枸杞岛附近海域表层水中的真菌群落组成特点,为枸杞岛附近海域表层水中微生物资源的开发利用及环境保护提供参考。     

除虫菊TcALDH和TcGLIP基因启动子克隆及功能分析

天然除虫菊酯是从除虫菊(Tanacetum cinerariifolium)中提取的绿色植物源生物杀虫剂。醛脱氢酶(TcALDH)和GDSL脂肪酶(TcGLIP)是除虫菊酯生物合成途径中的关键限速酶。为探究TcALDH和TcGLIP基因的功能,该研究从除虫菊无性系‘W99''中克隆得到TcALDH和TcGLIP基因的启动子,并通过生物信息学分析、组织化学染色(GUS染色)、荧光素酶报告实验和外源植物激素处理实验对其启动子的调控元件、启动子活性、激素诱导特异性和组织特异性进行分析。结果表明:(1)克隆得到的TcALDH和TcGLIP启动子序列分别为2 848、1 343 bp,均含有多个与逆境应答和激素信号相关的顺式作用元件。(2)分别构建了启动子和荧光素酶融合的植物表达载体,在烟草叶片中观察荧光成像发现,TcALDH启动子具有茉莉酸甲酯(MeJA)和脱落酸(ABA)激素诱导特异性。(3)用MeJA和ABA处理除虫菊‘W99''组培苗发现,TcALDH的表达量在12 h内受ABA诱导时上调,受MeJA诱导时先升高后降低,TcGLIP的表达量受ABA和MeJA诱导下调。(4)分别构建了TcALDH和TcGLIP启动子与GUS基因融合的植物表达载体,转化烟草并对其转基因叶片进行GUS活性染色发现,TcALDH启动子在烟草叶片腺体、腺毛头部及叶肉细胞中表达,而TcGLIP启动子仅在烟草叶肉细胞中表达。综上认为,TcALDH和TcGLIP的启动子具有组织特异性,TcALDH启动子具有MeJA和ABA激素诱导特性。该研究结果为除虫菊TcALDH和TcGLIP基因参与除虫菊酯合成的调控机制提供了新见解。     

喀斯特典型地区烟管荚蒾AM真菌多样性研究

烟管荚蒾是喀斯特地区灌木丛的主要组成植物。以茂兰喀斯特森林为采样地点,对烟管荚蒾AM真菌进行了分离、鉴定与多样性分析,并对优势菌种进行初步的接种研究。结果表明:烟管荚蒾根系的菌根侵染率为82.1%,从根际土壤分离到AM真菌2属16种,球囊霉属(Glomus)7种,无梗囊霉属(Acaulospora)9种。优势种为根内球囊霉(Glomus intraradices)、皱壁无梗囊霉(Acaulospora rugosa)和刺无梗囊霉(A. spinosa)。优势菌种接种紫花苜蓿(Medicago sativa),促生作用明显,并显著提高了SOD、POD和CAT酶活性。该研究对于探讨喀斯特地区AM真菌的多样性与独特性,筛选优良的宿主植物和与之高效共生的AM真菌具重要意义。     

2种杓兰属植物内生狭截盘多毛孢菌的分离与鉴定

该文报道了西藏杓兰(Cypripedium tibeticum)和无苞杓兰(C. bardolphianum)根中内生真菌新记录种——狭截盘多毛孢(Truncatella angustata)。该研究从四川黄龙沟不同海拔区分别采集到西藏杓兰和无苞杓兰,并在其根中分离获得148个狭截盘多毛孢菌株。形态学观察显示,狭截盘多毛孢菌株在PDA培养基上菌落白色,背面黄褐色;分生孢子器黑色脓包状;分生孢子梭型,4个细胞,3个隔膜;顶端附属丝1~5根。ITS rDNA分析发现3种新基因型菌株(HLIO15_15a_22、HLIO15_20a_42和HLIO15_17a_46);在海拔最高(3 330~3 400 m)的居群中狭截盘多毛孢的分离率最高,且包含全部3种基因型菌株。构建截盘多毛孢属(Truncatella)真菌ITS序列系统发育树,发现狭截盘多毛孢不同生态功能的菌株没有形成明显分支。     

葡萄生单轴霉菌围可培养微生物多样性及潜在生防菌研究

【目的】揭示葡萄生单轴霉(Plasmoparaviticola)菌围可培养细菌和真菌的多样性特征,筛选对葡萄霜霉病有较强稳定防治效果的生防菌。【方法】连续两年从我国南北方具有代表性的7个葡萄产区采集葡萄霜霉病叶,镊子夹取经保湿培养获得的新鲜霉层并配制孢子囊悬浮液,采用传统分离培养法,结合形态分类、BOX-PCR指纹图谱分析以及分子鉴定结果,对葡萄生单轴霉菌围的可培养细菌和真菌进行聚类分析;采用菌株及其发酵液与病原菌孢子囊悬浮液等体积混合培养测定其对孢子囊的抑制作用,离体叶片接种法检测该菌株及其发酵液对霜霉病的防治效果。【结果】分离获得了90株细菌和110株真菌,分别归属于8个细菌属和14个真菌属,且相同地区不同葡萄品种葡萄生单轴霉菌围的细菌和真菌在同年处于同一分支。假单胞菌属(Pseudomonas)和枝孢属(Cladosporium)稳定存在于各地区不同品种葡萄霜霉病叶上葡萄生单轴霉菌围;在两年间稳定存在的菌株占比多数在80.0%以上且均具有较高的生防作用;其中,广泛分布的6株枝顶孢属(Acremonium)真菌对葡萄霜霉病的防治效果均较好,最高可达100.0%;防治效果较高的11个菌株的无菌发酵液中,黑曲霉(Aspergillusniger) NX2F、苋楔孢黑粉菌(Thecaphora amaranthi) BJ1G和匍枝根霉(Rhizopus stolonifer) BM1L的无菌发酵液防治效果均为100.0%。【结论】葡萄生单轴霉菌围的可培养细菌和真菌群落主要受地区因素影响,有较高的稳定性和生防作用,揭示了枝顶孢属真菌在我国葡萄主要产区葡萄生单轴霉菌围附生的普遍性,为葡萄霜霉病的防治提供了丰富和宝贵的资源。     

福建省五种植物新记录

植物资源普查是掌握植物物种及数量变化的基础,对研究植物的地理分布和生物多样性具有重要意义。通过对2017年—2018年期间福建省本土植物调查过程中采集的植物标本进行整理和鉴定,发现了福建省被子植物5个新记录种,即台湾附地菜(Trigonotis formosana)、浙江琴柱草(Salvia nipponica subsp. zhejiangensis)、笔龙胆(Gentiana zollingeri)、大头茶(Polyspora axillaris)和钝颖落芒草(Piptatherum kuoi),均为福建省新记录种,大头茶属(Polyspora)和落芒草属(Piptatherum)为福建省新记录属。这些新记录植物的发现,丰富了福建省原生植物资源,为植物区系研究提供了新的资料。     

Cytoplasmic acidification as an early phosphorylation-dependent response of tobacco cells to elicitors

Elicitor-induced cytoplasmic pH changes of tobacco (Nicotiana tabacum L. cv. Xanthi) cells grown in suspension cultures were explored under a variety of conditions by using a flexible technique based on the distribution of [¹⁴C] benzoic acid between the intracellular and extracellular compartments. Comparison of data obtained by this technique and by ³¹P-nuclear magnetic resonance spectrometry qualifies the benzoic acid distribution method as a convenient and reliable way to probe cytoplasmic pH variations. Various elicitors shown to induce several defense-related responses in tobacco cells, namely oligogalacturonides of degree of polymerization 7–20, pectolyase from Aspergillus japonicus, Phytophthora megasperma crude elicitors and purified cryptogein, triggered cytoplasmic acidifications differing in intensity and kinetics according to the signal molecule. In contrast, no changes in cytoplasmic protons and external pH were observed in cells treated with short galacturonide oligomers, or with soybean-specific hepta -glucoside from P. megasperma, which are devoid of elicitor activity in tobacco cells. The oligogalacturonide-induced cytoplasmic acidification was inhibited by two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, which both reduced the external alkalinization response to the elicitor. The protein phosphatase inhibitor calyculin A alone behaved as an elicitormimicking molecule in triggering cytoplasmic acidification, again associated with extracellular alkalinization. These results indicate that the increase in the cytoplasmic concentration of protons may be considered as a common early intracellular response of tobacco cells to elicitors, associated with the extracellular alkalinization response and controlled by protein phosphorylation.Abbreviations BA(H) benzoic acid (protonated form) - 6-DMAP 6-dimethylaminopurine - DP degree of polymerization - Mes 2-(N-morpholino)ethanesulfonic acid - OG oligogalacturonide - pHc cytoplasmic pH - ³¹P-NMR nuclear magnetic resonance spectroscopy of ³¹P atoms The authors thank P. Albersheim (CCRC, Athens, Georgia, USA) for providing the purified oligogalacturonides and the hepta -glucoside and P. Ricci (INRA, Antibes, France) for providing the purified cryptogein.     

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28

Extracellular alkalinization and H₂O₂ production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H₂O₂ production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1–3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.     

Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells

Suspension-cultured cells of Lycopersicon peruvianum L. reacted to the presence of mechanically damaged cells with a transient alkalinization of their culture medium. This response resembled the alkalinization observed after treatment with fungal signal molecules such as chitin fragments and ergosterol or after application of the protein phosphatase inhibitor calyculin A. When compounds implicated in wound signalling were tested, the 18 amino acid peptide systemin was found to be a potent inducer of the alkalinization response, with a half-maximal activity at concentrations of ～100 pM. The decrease in extracellular H⁺ was paralleled by an increase of K⁺, and induction of both ion fluxes was blocked by the protein kinase inhibitor K-252a. Systemin also caused rapid increases in the activities of 1-aminocyclopropane-1-carboxylate (ACC) synthase and phenylalanine ammonia-lyase, two other responses commonly observed in cells treated with elicitors. The systemin analogue systemin-Ala¹⁷, a reported systemin antagonist in the induction of proteinase inhibitors in tomato plants, provoked a much weaker alkalinization response and did not induce ACC synthase at all. When applied together with authentic systemin, this analogue antagonized induction of both responses, indicating that the perception system for systemin had very similar properties in the L. peruvianum cells as in tomato plants. In conclusion, suspension-cultured L. peruvianum cells provide a convenient and highly sensitive system to study elements of wound response and, in particular, systemin perception.     

Early perception responses of<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> cells in response to lipopolysaccharides from<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis>

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe- / pathogen-associated molecular patterns, have diverse roles in plant–microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS_B.cep. was found to trigger a rapid influx of Ca²⁺ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K⁺/H⁺ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS_B.cep. elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS_B.cep. interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.Abbreviations DDC Diethyldithiocarbamate - DMSO Dimethyl sulfoxide - DPI Diphenylene iodonium - H ₂ DCF-DA 2,7-Dihydrodichlorofluorescein-diacetate - LPS Lipopolysaccharides - NAC N-Acetyl-l-cysteine - PTIO 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - ROS Reactive oxygen species - YE Yeast elicitor     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases

Studies of induced systemic resistance using strains of plant growth-promoting rhizobacteria (PGPR) have concentrated on the use of individual PGPR as inducers against multiple diseases of a single crop. To date, few reports have examined the potential of PGPR strain mixtures to induce systemic resistance against diseases of several different plant hosts. The objective of this study was to select mixtures of compatible PGPR strains with the capacity to elicit induced systemic resistance in four hosts. The specific diseases and hosts tested in this study included: bacterial wilt of tomato (Lycopersicon esculentum) caused by Ralstonia solanacearum, anthracnose of long cayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichum gloeosporioides, damping off of green kuang futsoi (Brassica chinensis var. parachinensis) caused by Rhizoctonia solani, and cucumber mosaic virus (CMV) on cucumber (Cucumis sativus). To examine compatibility, seven selected PGPR strains were individually tested for in vitro antibiosis against all other PGPR strains and against three of the tested pathogens (R. solanacearum, C. gloeosporioides, and R. solani). No in vitro antibiosis was observed among PGPR strains or against pathogens. Twenty-one combinations of PGPR and seven individual PGPR were tested in the greenhouse for induced resistance activity. Results indicated that four mixtures of PGPR and one individual strain treatment significantly reduced the severity of all four diseases compared to the nonbacterized control: 11 mixtures reduced CMV of cucumber, 16 mixtures reduced bacterial wilt of tomato, 18 mixtures reduced anthracnose of long cayenne pepper, and 7 mixtures reduced damping off of green kuang futsoi. Most mixtures of PGPR provided a greater disease suppression than individual PGPR strains. These results suggest that mixtures of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in the four hosts tested.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Active oxygen species production in tobacco cells elicited by cryptogein*

We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H₂O₂) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O₂^˙? abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H₂O₂ production through O₂^˙? dismutation. Although H₂O₂ production could be, per se, the origin of the pH changes observed, we showed that it was not the main cause, since DPI and Tiron did not inhibit extracellular alkalinization. On the other hand, cryptogein-induced changes in pH could be abolished using fusicoccin (FC), which is known to stimulate the plasmalemma H⁺ ATPase. Consequently, the observed changes in pH induced by cryptogein could be mainly due to the inhibition of the plasmalemma H⁺-ATPase activity. Furthermore, changes in extracellular pH were shown to modulate the intensity of AOS production by elicited cells. The possible regulation of the NAD(P)H oxidase activity of plant cells by changes in pH is further discussed.