The influence of growth conditions on biomass, toxins and pathogenicity of Fusarium oxysporum f. sp. orthoceras, a potential agent for broomrape biocontrol

The influence of different combinations of illumination and shaking on the growth dynamics, pathogenicity and toxin production of the fungus Fusarium oxysporum f. sp. orthoceras, a biocontrol agent of Orobanche cumana, was studied. The fastest biomass accumulation was obtained under shaking, with or without illumination, with the highest biomass obtained after 3–4 weeks of growth. The biological activity of chloroform extracts of the culture filtrate was characterised: it contained at least two main toxic metabolites that caused necrosis and wilting of various plants and led to mortality of germinating seeds of O. cernua, O. aegyptiaca, O. ramosa and O. cumana. The highest toxic activity of the chloroform extract was obtained under illumination without shaking after 3–4 weeks of growth. The two toxic metabolites were purified and identified as fusaric acid (FA) and 9,10‐dehydrofusaric acid (DFA). Both FA and DFA production began in the first week of growth, increasing gradually to their maxima after 4 weeks. The highest level of pathogenic activity of the fungus was obtained after three or more weeks of fungal growth. It can be concluded that in order to produce high levels of toxin and pathogenic activity, the fungus should be grown under illumination without shaking for 4 weeks.     

棉花枯萎病菌镰刀菌酸的产生和致病力的关系

 镰刀菌酸是棉花枯萎病菌的次生代谢产物,对一种非特异性的毒素。为了探索棉花枯萎病原菌的镰刀菌酸产量与致病力的关系及不同棉花品种对镰刀菌酸的反应,本实验测定了10个新疆吐鲁番地区的菌株和20个内地的菌株,发现其产镰刀菌酸的数量多少与生理型无关.培养初期各菌株的镰刀菌酸产量与菌株的致病力有关.陆地棉的10个品种对镰刀菌酸的抗性与它们在生产中表现出对枯萎病的抗性一致.     

Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.     

Zinc Improves Biocontrol of Fusarium Crown and Root Rot of Tomato by Pseudomonas fluorescens and Represses the Production of Pathogen Metabolites Inhibitory to Bacterial Antibiotic Biosynthesis

ABSTRACT Crown and root rot of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici is an increasing problem in Europe, Israel, Japan, and North America. The biocontrol agent Pseudomonas fluorescens strain CHA0 provides only moderate control of this disease. A one-time amendment of zinc EDTA at 33 mug of Zn(2+)/ml to hydroponic nutrient solution in soilless rockwool culture did not reduce disease when used alone, but did reduce disease by 25% in the presence of CHA0. In in vitro studies with the pathogen, zinc at concentrations as low as 10 mug/ml abolished production of the phytotoxin fusaric acid, a Fusarium pathogenicity factor, and increased production of microconidia over 100-fold, but reduced total biomass. Copper EDTA at 33 mug of Cu(2+)/ml had a similar effect as zinc on the pathogen in vitro; it reduced disease when used alone, and increased the biocontrol activity of CHA0 in soilless culture. Ammonium-molybdate neither improved the biocontrol activity of CHA0 nor affected production of fusaric acid or microconidia. Strain CHA0 did not degrade fusaric acid. Fusaric acid at concentrations as low as 0.12 mug/ml repressed production by CHA0 of the antibiotic 2,4-diacetylphloroglucinol, a key factor in the biocontrol activity of this strain. Production of pyoluteorin by CHA0 was also reduced, but production of hydrogen cyanide and protease was not affected, suggesting that fusaric acid affects biosynthesis at a regulatory level downstream of gacA and apdA genes. Fusaric acid did not affect the recovery of preformed antibiotics nor did it affect bacterial growth even at concentrations as high as 200 mug/ml. When microbial meta-bolite production was measured in the rockwool bioassay, zinc amendments reduced fusaric acid production and enhanced 2,4-diacetylphloro-glucinol production. We suggest that zinc, which did not alleviate the repression of antibiotic biosynthesis by fusaric acid, improved biocontrol activity by reducing fusaric acid production by the pathogen, which resulted in increased antibiotic production by the biocontrol agent. This demonstrates that pathogens can have a direct negative impact on the mechanism(s) of biocontrol agents.     

枯萎病菌毒素培养滤液对唐菖蒲幼苗毒性的初步研究

 对唐菖蒲枯萎病菌进行了产毒培养基的筛选,研究毒素培养滤液对胚根生长的抑制作用、对幼苗根系活力的影响及对幼苗的致萎作用。结果表明：唐菖蒲枯萎病菌在Czapek培养基中培养15 d所产生的培养滤液对唐菖蒲胚根的抑制作用最强,胚根生长抑制率高达99.79%;而菌丝干重在PD培养基中培养13 d达到最高值1 242.27 mg;毒素培养滤液经121℃灭菌20 min后对唐菖蒲胚根仍有很强的抑制作用,表现出较高的热稳定性;与对照相比,毒素培养滤液能够显著降低唐菖蒲幼苗根系活力,随处理时间的延长和处理浓度的增加,萎蔫级数逐渐增加。说明枯萎病菌毒素培养滤液是唐菖蒲枯萎病的致病因子,可以利用其筛选唐菖蒲抗枯萎病品种。     

The basis of host recognition in Fusarium oxysporum f. sp. lycopersici

Filtrates from shake-cultures of Fusarium oxysporum f. sp. lycopersici race 1, concentrated to 20% of the original volume, caused cell death in tomato leaf protoplasts from near-isogenic lines corresponding to the compatible cultivar/race reactions of whole plants. Maximum activity was found in late log phase cultures on Czapek-Dox supplemented with 2% casamino acids. Selective toxicity was associated only with the protein fraction of the culture filtrate. LD₅₀ values for susceptible Ace and Moneycross to F. oxysporum f. sp. lycopersici race 1 culture filtrates were 1·92 and 0·36 μg protein ml⁻¹. Corresponding values for cvs Royal Ace and MM161, each containing the I-gene conferring resistance to race 1, were >350. Culture filtrates from F. oxysporum f. sp. lycopersici race 2 gave LD₅₀ values of 2·34 and 2·08 μg protein ml⁻¹ on cvs Ace and Royal Ace, both susceptible to race 2. The LD⁵⁰ of cv. Ace to a non-pathogenic isolate of F. xysporum f. sp. lycopersici was > 350. Culture filtrates from non-host formae of F. oxysporum were 9–149-fold less toxic on cv. Ace. Protoplasts from Pisum sativum, Lactuca sativa, Zea mays, Gossypium barbadense and Solanum melongena, all non-hosts of F. oxysporum f. sp. lycopersici, were 6–175 times less sensitive to F. oxysporum f. sp. lycopersici filtrates than susceptible tomato. The putative toxins lycomarasmin and fusaric acid showed no differential toxicity to I⁺ and I⁻ tomato protoplasts. The results are discussed in the wider context of host-pathogen interaction in which specificity is considered as the recognition of susceptibility by a proteinaceous toxic metabolite of the pathogen. This hypothesis is further extended to include the specificity of F. oxysporum formae and races.     

Role of lipid metabolism through bioremediation of fusaric acid in germinating peanut seedlings

The effect ofBacillus subtilis on the contents of total lipids, neutral lipids, phospholipids and fatty acids in cotyledonary leaves of peanut (Arachis hypogaea L. cv. ‘Giza 6’) was studied in the presence and absence of the mycotoxin fusaric acid. In experiments conducted in a water culture in test tubes, the amount of total lipids was decreased by fusaric acid and increased byB. subtilis; combined treatment reduced the amount. With respect to the effect of fusaric acid on neutral lipids, a non-significant increase in diacylglycerol, significant decreases in triacylglycerol and sterol, and significant increases in sterol ester and non-esterified fatty acids, were obtained, whereasB. subtilis had the opposite effect. Generally, the amounts of the detected phospholipid fractions and the percentages of unsaturation index as well as fatty acids (palmitic, stearic and oleic) were reduced by fusaric acid. Linoleic acid, on the other hand, showed a reverse response: it was increased by fusaric acid alone or in combination withB. subtilis, the increase in the first case being greater than in the second, whereas linoleic acid disappeared in theB. subtilis treatment. http://www.phytoparasitica.org posting Dec. 16, 2003.     

Compounds inhibitory to nematophagous fungi produced by Bacillus sp. strain H6 isolated from fungistatic soil

Soil fungistasis can cause inconsistent control by nematophagous fungi of plant-pathogenic nematodes in field situations. Recent studies have shown that production of fungistatic compounds by bacteria was the principal explanation for soil fungistasis. The culture filtrate of Bacillus sp. strain H6, a strain representative of the dominant colony types isolated from fungistatic soils, showed strong inhibitory activity against nematophagous fungi by inducing unusual swelling in the conidia and the germ tubes of nematophagous fungi, thereby preventing the fungi from proliferating. This inhibitory mechanism is novel in comparison with other known mechanisms. Antifungal activity of the culture filtrate of strain H6 was maximal after culture in Luria Bertani (LB) broth (pH 7.0) at 36°C for 36 h. The inhibitory effect of the compounds produced by Bacillus sp. strain H6 was not significantly influenced by pH, and the inhibitory compounds in the culture filtrate were thermostable. After being partially purified by thin layer chromatography (TLC) on silica gel plates, characterization by colour reactions and positive fast atom bombardment mass spectrometry indicated that the inhibitory compounds showed similarity to iturin A group compounds.     

Effects of fusaric acid on cells from tomato cultivars resistant or susceptible toFusarium oxysporum f. sp.Lycopersici

Cell suspension cultures were set up from two tomato cultivars, one resistant, (Rio grande) and one susceptible (63.5) toFusarium oxysporum f. sp.lycopersici. Growth rates of the two cell cultures were comparable. Toxicity of fusaric acid, expressed as the fresh weight loss, was analyzed: It was significant in both cases after 10 h, but toxicity was twice as high for 63.5 suspension cells. In the same way, electrolyte leakage caused by fusaric acid was three times more important for 63.5 suspension cells. Moreover, fusaric acid treatment resulted in an acidification of the extracellular medium for 63.5 suspension cells (0.4 pH unit), whereas an alkalization was observed for Rio grande suspension cells (0.2 pH unit). Preliminary experiments suggest that fusaric acid was partially metabolized by Rio grande suspension cells, however, no detoxified forms of fusaric acid were detected either in cells or in culture filtrates. For these two tomato cultivars, the differences in sensitivity to fusaric acid of cultivated cells correspond to the differences in plant susceptibility toFusarium oxysporum f. sp.lycopersici.Abbreviations BAP 6-benzylaminopurine - conductivity - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOAc ethyl acetate - FA fusaric acid - resistivity     

青稞散黑穗病拮抗菌LN-176的分离鉴定、发酵条件及发酵产物的研究

从西藏佩古错湖湖边的土壤中分离到一株对青稞散黑穗病菌具有较强抑制作用的细菌,经鉴定为多粘芽孢杆菌,命名为LN-176。该菌株对多种植物病原真菌、部分革兰氏阳性细菌及革兰氏阴性细菌有较好的拮抗作用。以10%的接种量将该菌接种到初始pH7.0的发酵培养基中,28℃旋转培养,发酵液在84h时抗菌活性达到最高,且具有较好的热稳定性和pH稳定性,对蛋白酶K有一定的耐受性。LN-176菌株的发酵液在260nm和280nm下进行吸光度测定,结果表明发酵液中蛋白质含量明显升高;发酵液经硫酸铵盐析后,取上清液和沉淀物分别做抗菌试验,发现沉淀物对青稞散黑穗病菌有拮抗活性而上清液无活性。     

Differential effects of phytotoxic metabolites from <Emphasis Type="Italic">Alternaria tagetica</Emphasis> on <Emphasis Type="Italic">Tagetes erecta</Emphasis> cell cultures

Alternaria tagetica, a fungus that causes early blight in marigold (Tagetes erecta), produces two groups of phytotoxic metabolites: one hydrophilic and the other lipophilic that show phytotoxic activity when tested by the leaf-spot assay in T. erecta. We evaluated the cellular effects of the phytotoxic culture filtrate of A. tagetica and the hydrophilic and lipophilic fractions, then determined whether the filtrate or the fractions differentially induced pathogenesis-related mechanisms in the plant. The culture filtrate and the phytotoxic fractions had adverse effects on cell viability, fresh mass, and the number of cells, and induced ROS accumulation, lipid peroxidation and DNA damage on T. erecta cell suspension cultures, and these effects are related to pathogenic mechanisms attributed to phytotoxins. However, although exposure of marigold cells to the phytotoxic culture filtrate of A. tagetica triggered programmed cell death, the hydrophilic and the lipophilic phytotoxic fractions induced death that was more related to a toxic effect leading to necrosis. This study presents a complementary perspective in the search for the roles of metabolites, including phytotoxins, produced by phytopathogenic fungi during plant infection.     

生防链霉菌JH108-2的发酵培养基优化

 为了提高链霉菌菌株JH108-2的发酵滤液对植物寄生线虫的毒力,采用正交试验设计,优化其发酵培养基。使用优化培养基[大豆粉20g、(NH₄)₂SO₄ 6g、蛋白胨3g、葡萄糖40g、酵母粉6g,NaCl 2g、K₂HPO₄ 0.6g.CaCO₃ 2g、蒸馏水1000mL、pH 6.0]所得的发酵滤液,稀释10倍,处理南方根结线虫(Meloidogyne incognita)二龄幼虫,24h后校正死亡率达95.4%,比对照培养基的发酵滤液所产生的校正死亡率高6.9%,差异达到极显著水平(P=0.01)。     

玉米大斑病菌特异性毒素组分的分离与纯化

 玉米大斑病菌的2号小种菌株用改良Fries培养液进行体外培养,培养滤液经-40℃冷冻干燥,加等体积甲醇去除沉淀后用乙酸乙酯提取,粗提物经HPLC C₁₈柱可以分离出8个组分(峰3~10)。经生物测定发现,7号峰和10号峰的纯品对玉米叶片有明显的毒性,其中7号峰对带有Ht1基因的玉米表现出了一定的特异性。制备所得毒性组分分别进行红外光谱扫描,发现2个毒性组分的红外吸收光谱基本一致,吸收峰形状基本相同,只是其波数稍有变动。     

Purification and partial characterization of xylanase from the fungal maize pathogenHelminthosporium turcicum (Pass)

The fungal pathogenHelminthosporium turcicum was found to secret xylanase when grown on minimal medium containing xylans, wheat straw or isolated maize cell walls. The highest xylanase activity occurre when the fungus was grown on maize cell walls. When glucose was added to this medium xylanase activity was suppressed. The xylanase enzyme was purified from the culture filtrate by subsequent anion exchange chromatography, cation exchange chromatography and gel filtration. The purified xylanase gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to an apparent molecular weight of 22.5 kDa. It is determined to have a pI of 7.4, specific activity of 11300 nanokatals mg^–1, pH optimum between pH 5.5 and 6.5 and optimal temperature between 50 °C and 60 °C. The half-life of the enzyme at pH 6.0 and 50 °C was found to be 35 min. For primary structure comparison with other xylanases, the protein was digested with trypsin and the resulting peptides were separated by reversed phase chromatography and selected peptides were sequenced. The determined amino acid sequence showed high homology with xylanase fromCochliobolus carbonum and three other fungal xylanases.     

镰刀菌酸在尖孢镰刀菌侵染过程中的产生规律及运输过程探究

 镰刀菌酸(FSA)是尖孢镰刀菌产生的主要毒素之一,对病害的发展起到主导作用,但是其产生及运输机制尚不清楚,明确镰刀菌酸在寄主体内的产生过程及运输方式对防控作物枯萎病的发生具有重要的理论意义。本研究以黄瓜品种津春4号和尖孢镰刀菌黄瓜专化型菌 (Fusarium oxysporum f. sp. cucumerinum) 为试材,进行温室营养液培养试验,对不同侵染时期植株体内的病原菌及镰刀菌酸进行定量分析,结合韧皮部烫伤及分根根盒装置,探究镰刀菌酸的运输及分配机制。结果表明,病原菌的侵染首先从根尖或者侧根原基侵入根系,随后侵入维管组织并局限在木质部导管中,快速大量繁殖并开始产生毒素镰刀菌酸,镰刀菌酸主要通过木质部运输到叶片,发病期(11 dpi)叶片中镰刀菌酸含量是根系中镰刀菌酸含量的10倍。病原菌主要借助镰刀菌酸加速植物萎蔫死亡,并且镰刀菌酸含量与病原菌数量的相关性关系呈显著正相关。本研究进一步明确了病原菌侵染与镰刀菌酸产生的关系,病原菌在根系定殖成功后快速繁殖进入潜育期,随后产生毒素镰刀菌酸,并通过木质部运往地上部,促进叶片萎蔫,加速病原菌进入腐生阶段。在实际生产中我们可以在病害发生前期,未产生大量镰刀菌酸之前加强田间管理,以抑制或减轻枯萎病的发生。     

Auxin‐mediated relationships between apple plants and root inhabiting fungi: impact on root pathogens and potentialities of growth‐promoting populations

This study aimed to elucidate the relationship between plant hosts and root‐colonizing fungi recovered from apple orchard soils that had been replanted over multiple generations. Functional relationships of three groups of filamentous fungi (Ceratobasidium sp., Cylindrocarpon‐like group and Fusarium acuminatum) with apple rootstocks were evaluated in plant growth bioassays. The Cylindrocarpon‐like group and Ceratobasidium sp. showed a relationship with the host plant varying from pathogenic to commensal through to mutualistic for the latter group, while that of F. acuminatum tended to be mutualistic. Seven fungal isolates of each group, which induced the highest plant growth in bioassays, were evaluated for auxin (IAA) and gibberellin (GA₃ and GA₄) production in culture filtrate. All isolates of F. acuminatum as well as most of those of the Ceratobasidium sp. and Cylindrocarpon‐like groups produced IAA in culture filtrate. IAA production was evaluated for additional isolates of endophytic fungal species from fruit tree orchards and the functionality of IAA was confirmed by growing in vitro micropropagated plantlets of apple rootstock on MS medium supplemented with fungal culture filtrate. Findings from this study may explain the difficulty in defining the precise role of diverse root‐colonizing fungal populations in replant disease aetiology of fruit tree orchards. However, the results demonstrate the presence of a positive and widely available biotic component of the orchard soil biology that may be exploited for the benefit of tree growth and production.     

Fusarium verticillioides as a new pathogen of the parasitic weed Orobanche spp.

A strain of Fusarium verticillioides was isolated from the tubercles of the parasitic weed Orobanche cumana in Israel. The pathogenicity was tested in polyethylene bags on O. cumana, O. crenata, O. aegyptiaca and O. ramosa and in pots on O. cumana and O. crenata. F. verticillioides was highly pathogenic to O. aegyptiaca, O. ramosa and O. cumana in the polyethylene bags. In pots, the fungus caused wilting and necrotic areas on flowering shoots of O. cumana, but did not cause disease symptoms on O. crenata. F. verticillioides grown on liquid Czapek growth medium produced a phytotoxic metabolite, which in leaf-puncture bioassay caused large necrotic areas on Orobanche shoots and on leaves of various crops. An extract of the fungal growth medium caused complete mortality of O. cumana and O. aegyptiaca seedlings in vitro. The toxic metabolite was isolated and identified by spectroscopic methods as fusaric acid. The identity of the compound was confirmed by conversion into the corresponding methyl ester, and by TLC comparison against authentic fusaric acid. No other phytotoxic metabolites could be detected in the growth medium extracts.     

高毒力杀虫细菌嗜线虫致病杆菌CB6菌株的培养基优化

用正交试验法研究了嗜线虫致病杆菌北京变种菌株CB6的营养利用以及培养基成分的改变对产生杀虫活性物质的影响。通过筛选该菌对碳源、氮源以及无机盐的需求,确定了该菌的最佳发酵培养基为(g/L):豆粉40g,蔗糖15g,蛋白胨15g,酵母膏10g,玉米浆5g,NaH2PO41g,NaCl5g,谷氨酸5g。培养液上清液对玉米螟初孵幼虫的体重抑制率为90 81%,比基础培养基的杀虫活性提高了46.5%。     

Fusaric Acid Production by Fusarium Oxysporum f.sp. lilii and its Role in the Lily Basal Rot Disease

Fusarium oxysporum f.sp. lilii (Fol), the causal agent of lily basal rot, produced fusaric acid (FA) in aseptic culture. This toxin induced phytotoxicity symptoms on in vitro-grown lily bulblets of two different cultivars: the Fol-susceptible cultivar was more sensitive to the toxin than the Fol-resistant cultivar. When cultured in the presence of FA, the Fol-susceptible cultivar showed a greater tendency to accumulate FA within its tissues than the Fol-resistant cultivar. The polyphenol oxidase activity of the bulblets was inhibited by 1mmolL^–1 FA in both the cultivars, while at lower FA concentrations the enzyme activity increased only in the Fol-susceptible cultivar. Peroxidase showed a steady activity at the 1mmolL^–1 FA concentration in both the cultivars, while at lower FA dosages its activity increased. Within the Fol-infected in vivo tissues of both the lily cultivars, FA was detectable only in traces. The role of this toxin in the lily basal rot disease seems therefore to be of marginal importance.     

In vitro Effect of the Phytoalexin Glyceollin I on Polygalacturonase and Oxalic Acid Production ofSclerotinia sclerotiorum

The polygalacturonase (PG) activity in culture filtrates fromSclerotinia sclerotiorum is reduced when glyceollin I, the major soybean phytoalexin, is present in the culture medium. When the enzyme activities in the culture filtrates are expressed per unit of fungal growth, PG activity decreases with increasing concentration of glyceollin I in the culture medium. The phytoalexin does not influence the isoenzyme pattern. This suggests that glyceollin I may inhibit quantitative but not qualitative enzyme production. Only the highest glyceollin I concentration tested inhibits oxalic acid production. The inhibitory effect on mycelial growth is confirmed. The data suggest a further hypothesis about the role of phytoalexin during pathogenesis.