Screening Isolates of Hirsutella Species for Biocontrol of Heterodera glycines

The fungi, Hirsutella rhossiliensis and Hirsutella minnesotensis, are two endoparasites of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines. The objective of this study was to screen for effective fungal isolates to control the nematode in laboratory and greenhouse assays. A total of 93 isolates of H. rhossiliensis and 25 isolates of H. minnesotensis were evaluated for parasitism of SCN J2 on cornmeal agar. Percentage of SCN J2 parasitized by the fungi varied among the fungal isolates. Most H. rhossiliensis isolates parasitized a high percentage of J2. The isolates of H. rhossiliensis obtained from bacteria-feeding nematodes, however, generally did not parasitize J2 on agar. H. minnesotensis parasitized fewer J2 on agar than did H. rhossiliensis . Forty isolates of H. rhossiliensis and four isolates of H. minnesotensis that parasitized a high percentage of J2 on agar were evaluated for their biocontrol potential in soil treated with microwave heating. Most isolates selected from the agar assay also parasitized a high percentage of J2 in the soil but there was variation among isolates. Pathogenicity of 14 isolates of H. rhossiliensis and four isolates of H. minnesotensis to the SCN was also investigated in the greenhouse using untreated field soil. Most isolates of H. rhossiliensis reduced SCN population density and increased plant growth when compared with 1% corn-grits control (culture media). One isolate (OWVT-1) of H. rhossiliensis reduced the SCN egg density by 95% and J2 density by 98% when compared with the control. Isolates of H. minnesotensis, however, neither reduced SCN density nor increased plant growth in the greenhouse.     

Nutritional requirements of Pochonia chlamydospora and ARF18, fungal parasites of nematode eggs

Twenty carbohydrates (C), 18 nitrogen compounds (N), and 9 vitamins were examined for their effects on the growth and conidiation of the nematode-egg-parasitic fungi Arkansas Fungus 18 (ARF18, isolate 908) and Pochonia chlamydospora var. chlamydospora in solid and liquid cultures. Glycogen was the best, and inulin, D-(+)-galactose, and soluble starch were good C sources for the growth of ARF18 in both solid and liquid cultures. ARF18 could not utilize alpha-cellulose in liquid; alpha-lactose and D-mannitol in solid; and D-(+)-xylose, L-(-)-sorbose, and D-(-)-ribose in both solid and liquid cultures. Casein was the best N source for ARF18 growth in both solid and liquid cultures and L-aspartic acid, DL-glutamic acid, peptone, and L-histidine were good in solid culture. ARF18 could not utilize L-cystine and L-tyrosine in solid culture, and L-cystine, DL-methionine, peptone, L-proline, and ammonium nitrate in liquid culture. Supplement of vitamins appeared to be unnecessary for ARF18 to grow. For P. chlamydospora var. chlamydospora growth, all test C sources, except L-(-)-sorbose, alpha-cellulose, citric acid, and D-(+)-glucose, were good in both solid and liquid cultures. Most N compounds were good for P. chlamydospora var. chlamydospora growth with casein and peptone the best. Vitamins had limited effect on P. chlamydospora var. chlamydospora growth. P. chlamydospora var. chlamydospora conidiation was well supported by D-(-)-ribose, D-(-)-fructose, melibiose, and D-(+)-galactose as C sources and by L-aspartic acid, DL-glutamic acid, and L-arginine as N sources. Excluding myo-inositol from the medium containing all other test vitamins increased P. chlamydospora var. chlamydospora conidiation, while excluding pyridoxine appeared to reduce its conidiation.     

Cocoa-based media for culturing Phytophthora palmivora (Butl.) Butl., causal agent of black pod disease of cocoa

Green cocoa pod husk agar (GCPA), ripe cocoa pod husk agar (RCPA), green cocoabean agar (GCBA), ripe cocoa bean agar (RCBA), green cocoa mucilage agar (GCMA)and ripe cocoa mucilage agar (RCMA) were prepared and assessd for their clarity andfor potential to support mycelial growth and sporulation of P. palmivora. Oatmeal agar (OMA), potato-dextrose agar (PDA), vegetable 8 juice agar (V8JA) and pineapple crown agar (PCA) were included for comparison. The highest radial growth rates of 8.3 and 7.2 mm/day were recorded, respectively, on OMA and GCPA but these were not significantly different (P ≤ 0 05) from each other. The two media also supported good aerial mycelial growth but were not clear. Radial mycelial growth rates of 6.5, 7.0 and 6.6 mm/day were obtained on GCMA, RCPA and V8JA, respectively, and these rates were also not significantly different from each other. Of the three media, only the GCMA was clear and supported the best aerial mycelial growth. In comparison, the RCMA supported a significantly lower radial growth (4.6 mm/day) of P. palmivora than the three media. Growth rates were least on RCBA, PCA and PDA but sporulation was poorest on PDA, PCA and V8JA. GCMA was found to be the best medium based on all the growth parameters and media characteristics. GCMA has been used effectively to isolate/detect P. palmivora from infected cocoa pod tissues. Apart from differences in radial growth rate, both the GCMA and RCMA were similar in all other respects and are recommended for culturing P. palmivora. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts

Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.     

阔鳞鳞毛蕨孢子的无菌繁殖

以阔鳞鳞毛蕨（Dryopteris championii）成熟孢子为外植体,研究了不同植物生长调节剂及浓度对其孢子萌发、愈伤组织诱导、丛生芽分化及生根的影响。结果表明：1/2MS＋2%蔗糖为孢子萌发最适培养基,20 d后萌发率达65%;诱导愈伤组织的最适培养基为MS＋KT 0.5 mg.L-1＋2,4-D 1 mg.L-1,诱导率达39.8%,愈伤组织为绿色颗粒状;颗粒状愈伤组织在MS＋KT0.2 mg.L-1的培养基中生长出大量丛生芽,转化率可达66.3%;MS＋IAA 0.2 mg.L-1＋NAA 0.2 mg.L-1培养基可有效促进幼孢子体苗生根。     

Sporulation of Bacillus stearothermophilus

A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (NCA 1518) and four isolates identified as B. stearothermophilus. Maximal spore yields were obtained by use of inocula grown anaerobically in a medium containing glucose with aeration of sporulation medium via bubbling. After an extended stationary period, sporulation occurred concurrently with vegetative growth between 6 and 8 hr of incubation at 60 C. Omission of glucose from the inoculum or use of a “young” (2 hr) inoculum abolished the stationary period, but decreased spore yields. A requirement of oxygen for rapid vegetative growth and sporulation was demonstrated. Manganese (15 to 30 ppm) stimulated sporulation but did not enhance cell growth.     

Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g⁻¹ carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 10⁸ spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L⁻¹) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L⁻¹ carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production. Received 9 December 1997/ Accepted in revised form 22 May 1998     

食线虫真菌洛斯里被毛孢线粒体基因组的再分析

【目的】鉴定洛斯里被毛孢OWVT-1菌株的线粒体基因组,验证公布的USA-87-5菌株线粒体基因组中的错误,对洛斯里被毛孢正确的线粒体基因组序列进行注释并开展不同被毛孢物种间的比较线粒体基因组学分析。【方法】借助DNA高通量测序数据并通过必要的Sanger测序组装OWVT-1的线粒体基因组。通过PCR验证OWVT-1与公布的USA-87-5线粒体基因组序列差异的真实性。利用多种生物信息方法分析和注释洛斯里被毛孢的线粒体基因组。【结果】公布的洛斯里被毛孢USA-87-5菌株的线粒体基因组存在几处序列错误,包括3处长片段的插入缺失和多处短片段的插入缺失。实际上,洛斯里被毛孢USA-87-5与OWVT-1菌株的线粒体基因组序列完全相同。该菌的线粒体基因组全长62949 bp,在7个基因中共插入13个内含子,部分内含子和基因间区显现出序列退化的特征。洛斯里被毛孢、明尼苏达被毛孢、线虫被毛孢的线粒体基因组具有较强的共线性关系。除一些独立的ORF外,核心蛋白编码基因、rRNA基因和tRNA基因的排列顺序非常保守。基因间区的长短是影响3种被毛孢线粒体基因组大小最主要的因素。【结论】公布的洛斯里被毛孢USA-87-5菌株线粒体基因组中存在序列错误。本文新报道了OWVT-1菌株的线粒体基因组,并进行注释和比较线粒体基因组学分析。     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性。将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤, 分离得到脂肽类抗生素的纯品。经IC50实验和抗菌谱测定, 考察了脂肽类抗生素对多种植物病原菌的作用, 确定了脂肽类抗生素的抗菌谱。深入研究表明, 枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质, 能够抑制灰霉病菌孢子的萌发和菌丝的生长。脂肽类抗生素和挥发性抑菌物质的协同作用, 有助于提高枯草芽孢杆菌的生物防治效果。     

Germination, Radial Growth, and Sporulation of Beauveria bassiana and Metarhizium anisopliae Isolates and Their Virulence to Chilo partellus (Lepidoptera: Pyralidae) at Different Temperatures

The effect of temperature regimes on conidia germination, radial growth and virulence of Beauveriabassiana and Metarhizium anisopliae against the spotted stalk borer Chilo partellus was investigated with Ethiopian isolates. Conidia germination, radial growth and sporulation of all isolates were retarded at 15 and 35°C. A suitable temperature range for the isolates was between 20 to 30°C. Conidia germination was less tolerant of low temperature (15°C) than radial growth. Radial growth and sporulation reacted differently to temperature. At both 25 and 30°C, all isolates induced 100% mortality to C. partellus larvae in six days. The LT₅₀ decreased for the isolates with increasing temperature.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

Jasmonic acid promotes division of fern protoplasts, elongation of rhizoids and early development of gametophytes

The effects of jasmonic acid (JA) on spore germination, early gametophyte development and sporophytic protoplast culture of the fern Platycerium bifurcalum (Cav.) C. Chr. were investigated. JA and no influence on spore germination and primary rhizoid initiation, but significantly promoted early gametophyte development, which was evident from the longer primary rhizoids as well as the higher number of rhizoids and cells per gametophyte. Jasmonic acid (1 μ M ) also promoted the transition of gametophytes from a filamentous to a planar growth. Optimal primary rhizoid elongation and highest cell division activities in the gametophytes were observed at 0.01–1 μ M JA, while the highest number of rhizoids on gametophytes was obtained at 0.1–1 μ M JA. Jasmonic acid (0.01 μ M ) also stimulated initial protoplast divisions. Except for the experiment in which the effect of JA on germination was tested. JA concentrations exceeding 1 μ M did not promote cell elongation or cell division but were instead inhibitory. On the basis of these findings, we propose that JA may be involved in early stages of P. bifurcatum development.     

Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates

On agar media the maximum conidia production of Exserohilum monoceras occurred on V-8 juice agar (VA) or centrifuged V-8 juice agar, whereas the optimal radial mycelial growth occurred on Czapek-Dox agar. The optimal temperatures for radial mycelial growth and conidia production were 28 and 27°C respectively. Light prohibited E. monoceras conidia production. The best sporulation occurred under continuous dark conditions. Echinochloa leaf decoction significantly increased conidia production on potato dextrose agar (PDA) and VA, and significantly increased germ tube length on PDA, lima bean agar and VA, but did not affect conidia germination. No conidia were produced in liquid media. Of 22 agricultural-based products evaluated as solid substrates, the most abundant sporulation (1.8 × 10⁶ conidia g^-1 of dry weight) occurred on corn leaves. The conidia production of E. monoceras on corn leaves was affected by incubation period, moisture content and substrate quantity. There were no differences in germination rate, germ tube length and virulence of conidia produced on agar media or corn leaves.     

Role of plant growth regulators in host-pathogen relationships

The effect of indol-3-ylacetic acid, gibberellie acid, kinetin, abscisic acid and Ethrel on the growth of mycelium, sporulation and germination of spores ofFusarium culmorum of different pathogenicity to wheat seedlings was studied. The production of gibberellins, auxins, cytokinins, ethylene and growth inhibitors by these isolates was determined as well. It has been found that most pronounced and explicit effect on growth and development in fungi was produced by Ethrel which strongly inhibited these processes. ABA proved to be a strong growth and development stimulator, though to a different extent in different isolates. GA₃ strongly stimulated sporulation and spore germination in some isolates. The effect of IAA and K on growth and development in fungi was slight. More sensitive to growth regulators were the fungi in earlier stages of growth. No correlation between the pathogenicity of the isolates and their ability to produce growth regulators as well as between their susceptibility to exogenous growth substances in the processes of fungal growth and development was stated. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

草坪草离蠕孢叶枯病菌生物学特性的研究

对草坪离蠕孢叶枯病病原菌进行分离鉴定,并对其生物学特性进行了研究。结果表明:该病害由禾草离蠕孢(Bipolaris sorokiniana)引起。该病原菌的菌丝生长及产孢的最适温度为30℃,孢子萌发最适温度为25℃,菌丝的致死温度为65℃,而孢子的致死温度则为55℃;该菌对酸碱度的适应能力较强,中性偏酸性的条件对菌丝的生长有利,而pH值为8.0时最易产孢;各碳源对菌丝的生长均有促进作用,但不同碳源对产孢量的影响很大,单糖和双糖利于产孢,多糖对产孢的影响不大。氮源对菌丝生长和产孢量非常重要,无机氮效果较好,硝态氮好于氨态氮,有机氮效果最差。花粉、叶面物质和草坪草汁液可促进孢子萌发。     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

环境因素对香蕉叶斑病菌喙突脐蠕孢生长和产孢的影响

为进一步明确香蕉叶斑病菌喙突脐蠕孢的生物学特性,调查了部分环境因素(碳氮源、植物成分培养基)对供试菌株(CLER09、D087和JL05)的营养生长和产孢的影响。结果显示,分生孢子主要先从基部萌芽,在28℃下约培养10h后出现两端萌芽;供试菌株对测试的20种碳源及26种氮源显示相似的生长反应,均可利用除菊糖外的测试碳源及氮源进行营养生长和产孢;测试的6种植物成分培养基对供试菌株的营养生长效果皆优于PDA培养基;除燕麦培养基外,其余5种培养基对菌株CLER09和D087的产孢作用均优于PDA培养基;测试的6种培养基对菌株JL05的产孢作用均优于PDA培养基。不同植物成分培养基对该菌分生孢子形态影响较大,以米糠、象草培养基对菌株D087和JL05的分生孢子长度和玉米粉培养基对菌株CLER09的分生孢子宽度的增长效果最为明显。     

紫外、离子注入法对白腐真菌诱变效应的初步研究——多酚氧化酶菌株的选育

对白腐真菌F4孢子悬液进行紫外、N+离子注入诱变.诱变后待孢子长出单菌落,滴加茴香胺等多酚氧化酶底物,观察其颜色变化;经发酵筛选,获得一株多酚氧化酶高产菌POP5,漆酶活力是原出发菌株的16倍,并且得到一株多酚氧化酶缺失菌株POL1.紫外诱变,孢子浓度为106～108个/ml,照射时间1～2min;N+离子注入,孢子浓度为105～106个/ml,能量20Kev,剂量为5×1014ons/cm2,每个平板上生长30个左右菌落是最佳诱变选育条件.与其它真菌的孢子相比,N+离子注入法对白腐真菌F4孢子的致死率较大.