THE PRODUCTION OF ANTIBIOTICS IN SOIL: II. PRODUCTION OF GRISEOFULVIN BY PENICILLIUM NIGRICANS

A study has been made of the conditions affecting production of griseofulvin by Penicilliiim nigricans in two types of soil, an acid, sandy podsol from Wareham Heath and a garden soil. The characteristic morphogenetic response of many fungi to low concentrations of griseofulvin was made the basis of a highly specific bioassay.
The essential prerequisites for production of griseofulvin in either soil were sterilization and enrichment with organic matter; no griseofulvin could be detected in autoclaved soil which had not been supplemented or in normal soil even when organically enriched. Garden soil was a better medium for growth of P. nigricans and production of griseofulvin than Wareham soil although this soil could be improved in this respect by liming.
The yield of griseofulvin was decreased in soil re-infected by other soil organisms, particularly by some which were known to produce antifungal antibiotics, e.g. Penicilliunr expansum, P. frequentons and two strains of Trichoderma viride. The antagonism shown to Penicilliunz nigricuns was not entirely a matter of antibiotic activity, as some fungi believed not to produce antifungal substances had an antagonistic effect. These were mostly fungi with a characteristically rapid growth rate, e.g. Mucor rarnmannianus and one strain of Trichoderma riride. In some cases Penicillium nigricans was itself antagonistic to other fungi irrespective of their ability to produce antibiotics or of their fast-growing habit.
The results were compared with those obtained from a previous study of the soil conditions affecting the production of gliotoxin by Trichoderma viride. A higher level of nutrient was required for the production of griseofulvin, and the effect of antagonism by other soil micro-organisms was more important than in the production of gliotoxin by T. viride in the soil.     

Gliotoxin, a fungistatic metabolic product of Trichoderma viride

Gliotoxin is shown to be a metabolic product of Trichoderma viride , and a semi-continuous apparatus for its production is described. Ammonia nitrogen is preferable to nitrate nitrogen, but a wide range of carbon sources, and organic or inorganic sulphur sources, are suitable for gliotoxin production. No organic supplements to a glucose-mineral medium have been found to affect gliotoxin production beneficially.
Data are presented showing gliotoxin to be moderately toxic to a wide range of bacteria, actinomycetes and fungi. T. viride itself is resistant to its toxic effects. It shows fungicidal activity when applied as a dust to cereal seeds bearing various seed-borne diseases, but is inferior to organomercury compounds for this purpose. Its value as a fungicide for control of plant or animal infections is reduced by the instability of aqueous solutions, except at low p H.     

Biological control of a soil-borne pythium infection by seed inoculation

Summary 1. A three-fold effect was produced on white mustard seedlings grown in soil infected withPythium sp. Seed germination, the number of healthy plants which survived and the fresh weight of the shoots were reduced.2. Disease symptoms were controlled to some extent by dusting the seeds with spores of some common soil saprophytes includingTrichoderma viride, Penicillium nigricans, P. jrequentans andP. godlewskii.3. Of three strains ofTrichoderma viride which were tested for antagonism toPythium sp., a gliotoxin-producing strain was more effective in controlling the disease than a viridin-producing strain and an antibiotically inactive strain gave least protection to the seedlings.4. The disease symptoms were less severe in soil treated with acid or calcium hydroxide. Inoculation of the seeds withT. viride gave further control of the disease in soil treated with calcium hydroxide but not in acidified soils. These results are discussed in relation to the production of gliotoxin byT. viride.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL

Antibiotic production in and around particles of plant debris in soil was studied. High yields of an antibiotic, shown by bioassay methods to be similar to gliotoxin, were obtained from wheat straws buried in a normal, unautoclaved; acid podsoc from Wareham Heath which had been inoculated with a strain of Trichoderma viride known to produce gliotoxin in culture media. Only a little of the antibiotic was produced in the soil immediately surrounding the straws. Much less was produced in straws buried in John Innes potting compost and none at all in straws buried in a Kettering loam. In no case was an antibiotic detected in straws from un-inoculated soils.
If, however, the Kettering loam was acidified or, alternatively, the straws themselves were acidified and then buried in untreated Kettering loam, good yields of the antibiotic were obtained from straw extracts. Conversely, when the pH of Wareham Heath soil was raised by addition of calcium hydroxide to the soil no antibiotic activity could be detected in the straws. This suggests that the pH of the soil and of the food substrate has a profound effect on production of an antibiotic, assumed to be gliotoxin, by T. viride. The results obtained suggested that increased production of gliotoxin after autoclaving the straws was due to a decrease in the pH of the straws rather than to a release of nutrients.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL

Conditions affecting the production of gliotoxin by a strain of Trichoderma viride , known to produce this antibiotic in synthetic culture media, were studied in two types of soil, a highly acid, sandy podsol from Wareham Heath and a less acid garden soil. High yields of an antibiotic substance, which results from bioassays showed to be similar to gliotoxin, were obtained from both inoculated soils when autoclaved and supplemented with organic material. The autoclaved soils behaved differently when unsupplemented; Wareham soil supported production of the antibiotic but little or none was produced in the garden soil. No antibiotic activity could be demonstrated in soil which had not been inoculated with T. viride.
Acidification of unsupplemented garden soil by addition of sulphuric acid had a favourable effect on production of the antibiotic, but raising the pH of Wareham soil by addition of calcium hydroxide also increased the yield. These effects, therefore, cannot be due simply to the change in pH of the soil.
The beneficial effect of autoclaving the soil on production of the antibiotic assumed to be gliotoxin was analysed and separated into three distinct effects, elimination of the microflora, increase in availability of nitrogen compounds and increase in available carbon compounds. The last effect was considered to be of greatest significance.
The antibiotic was produced in normal Wareham soil if supplemented with additional carbon compounds, but not in garden soil unless this had also been acidified before inoculation. A chromatographic method of bioassay used in the later work gave more substantial evidence that the antibiotic produced in the soil was, in fact, gliotoxin.     

Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil.

Cultures of Sclerotium rolfsii and Trichoderma viride together in autoclaved soil were assayed at intervals during 8 days of incubation for proteolytic activity (PA) of T. viride. Significant proteolytic activity was detected only in soil containing T. viride (i.e., T. viride alone or S. rolfsii + T. viride); greatest activity occurred between 3 and 4 days after infestation and declined rapidly thereafter. Maximal PA in the mixed-culture soil was accompanied by an increase in soil pH. optimal pH values for PA was 5.5-6.5 with a maximum at 6.0.     

Effect on microorganisms of volatile compounds released from germinating seeds.

Volatile compounds evolved from germinating seeds of slash pine, bean, cabbage, corn, cucumber, and pea were evaluated for their ability to support growth of microorganisms in liquid mineral salts media lacking a carbon source. Growth of eight bacteria was measured turbidimetrically and of six fungi as dry weight of mycelium. Volatiles caused increased growth of Pseudomonas fluorescens, Bacillus cereus, Erwinia carotovora, Agrobacterium tumefaciens, A. radiobacter, Rhizobium japonicum, Mucor mucedo, Fusarium oxysporum f. conglutinans, Trichoderma viride, and Penicillium vermiculatum but not of Sarcina lutea, Serratia marcescens, Chaetomium globosum, or Schizophyllum commune. Spores of Trichoderma viride showed higher germination in the presence of volatiles. Effects on growth were apparent only during the first 3 or 4 days after planting the seeds. Killed or dried seeds had no effect. The volatiles did not support microbial growth in the absence of nitrogen nor did they supply growth factors. Passing volatiles through KMnO4 or hydrazone reduced growth of the bacteria, indicating that oxidizable organic compounds, primarily aldehydes, were the active components. The volatiles were not absorbed by sterile soil, clay minerals, or water, but they were absorbed by non-steril soil and activated charcoal.     

Seed positioning in extruded pellets: does it matter?

Extruded pellets containing activated carbon (AC) can be used to sow native seeds while simultaneously applying herbicide to control invasive species. Incorporating AC in pellets has been demonstrated to protect native seeds; however, there may be unintended detrimental impacts to seedling emergence. We aimed to optimize seed position within pellets to maximize emergence and survival of the perennial shrub Jacksonia furcellata. Seeds were positioned at 2 mm (top), 6 mm (middle), and 12 mm (bottom) within pellets (with or without AC), sown on or below the soil surface, and compared to non-pelleted seeds sown under the soil surface in the equivalent positions (2, 6, and 12 mm depth). Trays were treated with a pre-emergent herbicide (Simazine) or left unsprayed. Emergence (without herbicide) was significantly higher from seeds positioned at the bottom of pellets without AC sown on the soil surface (70%), compared to non-pelleted seeds sown at the bottom (12 mm below the soil surface; 57%). However, emergence was inhibited when seeds were positioned in the middle (6 mm) of pellets with AC (32%). When treated with Simazine, survival was highest from seeds positioned at the bottom of pellets with AC (60%), compared to pellets without AC (15%) and non-pelleted seeds sown at the bottom (12 mm below the soil surface; 15%). Jacksonia furcellata seeds positioned at the bottom of pellets, sown on the soil surface, shows promise to minimize negative impacts to emergence, and maximize herbicide protection. Further testing with additional species is required to refine pellet production (e.g. recipe, extrusion, and shape) for optimal emergence.     

Evaluation of Some Fungi and Bacteria for Potential Control of Safflower Rust

Some fungal and bacterial isolates applied as soil and seed treatments in greenhouse trials gave a good protection against Puccinia carthami Cda. on safflower, significantly reducing the infection from rust-infested seeds. Trichoderma viride and T. harzianum added alone and in combination as air-dried inoculum to the soil were the most effective fungal isolates. However T. viride at two higher rates reduced the rate of emergence of safflower seedlings. Among bacteria, Bacillus subtilis, B. cereus, isolates of Pseudomonas fluorescens and B. thuringiensis reduced rust infection when added both as soil drench and as seed treatment. Good results in the biocontrol of P. carthami were also obtained with some combination antagonist treatments such as T. viride + B. cereus, T. viride + P. fluorescens (16), T. viride + T. harzianum + B. cereus and T. viride +, T. harzianum + P. fluorescens (16). Fungal isolates added as soil treatment increased seedling length, whereas no similar effects were observed when these isolates were applied as seed treatment.     

木霉菌T23胶毒素合成基因的生物信息学分析与克隆

胶毒素是生防木霉菌重要的次生代谢产物之一。本研究以生防木霉菌T23为供试材料,旨在通过生物信息学技术及表达分析,挖掘木霉菌T23中胶毒素合成候选基因,探索木霉菌胶毒素合成的分子调控机制,可为新型生物农药的开发及应用提供理论依据。研究表明,木霉菌T23中胶毒素合成候选基因簇全长28 kb,簇内包含了8个基因,分别与烟曲霉胶毒素合成基因簇内的gliP、gliC、gliN、gliK、gliI、gliG、gliF、gliM高度同源。提取培养2 d、3 d、4 d、5 d的木霉菌T23菌丝的RNA,通过半定量RT-PCR技术探索各候选基因在木霉菌T23不同生长时期的表达情况,显示各基因在不同生长时期均有表达,属于组成型表达基因。成功克隆得到木霉菌T23中的gliP-T23基因并完成基因结构分析,该基因全长6 339 bp,由4个外显子和3个内含子组成,为后续的基因功能验证提供基础。     

豆薯种籽中一种抗真菌蛋白PaAFP的分离纯化和部分特性研究

各类植物由于缺少自身免疫系统的支持,因而必须依赖于其它机制来抵御外来微生物的入侵．其中的一种重要机制就是通过合成体内各类能抑制微生物生长的蛋白质来完成的［１］．已报道从植物中分离出多种不同的抗真菌蛋白．广为研究的是几丁质酶和β－１,３－葡聚糖酶,认为它们在植物对真菌病的抗性中起重要作用［２,３］;核糖体失活蛋白（ＲＩＰｓ）和一类富含半胱氨酸的碱性多肽Ｔｈｉｏｎｉｎｓ也显示有非专一的抗真菌活性［４,５］．但仍有一些蛋白质,体外表现强烈的抗真菌活性,却不属于以上范围［６,７］．本文报道了豆薯种籽中一…     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

鄂西北产苍耳子甲醇粗提物抑菌活性及其清除DPPH能力的初步评价

采用生长速率法、孢子萌发法及DPPH自由基清除法,对产自鄂西北的野生植物苍耳子粗提物体外抑菌活性及抗氧化性进行了初步测定.结果表明,苍耳子甲醇粗提物对绿色木霉、黄瓜灰霉菌、黑曲霉、终极腐霉、尖镰孢菌黄瓜专化型五种病原真菌均有一定的抑制作用,其中无论是抑制菌丝生长还是孢子萌发,均对黑曲霉显示出了显著的抑制活性.实验结果也...     

Effect of seed depth on slug damage to winter wheat

In a field experiment drilled at two depths on three dates in autumn 1988, with or without methiocarb pellets broadcast on the soil surface immediately after drilling, 26% of seeds of winter wheat sown at c. 20 mm depth were killed by slugs compared with only 9% of seeds sown at c. 40 mm. The protection from slug damage provided by this additional 20 mm of depth was comparable with that provided by methiocarb pellets. The effects of seed depth and pellet application did not interact and were consistent on all drilling dates. Thus, fewest seeds and seedlings were killed where methiocarb pellets were broadcast on a seed-bed with seeds sown at 40 mm depth. Intermediate damage was recorded where seeds were sown at 40 mm depth without pellets, or where pellets were broadcast on seeds sown at 20 mm depth. Most seeds and seedlings were killed where seeds were sown at 20 mm depth without pellets. Sublethal damage to seedlings was not affected by sowing depth but was reduced where pellets were broadcast immediately after sowing.     

Seed Dormancy,Seedling Establishment and Dynamics of the Soil Seed Bank of Stipa bungeana (Poaceae) on the Loess Plateau of Northwestern China

Studying seed dormancy and its consequent effect can provide important information for vegetation restoration and management. The present study investigated seed dormancy, seedling emergence and seed survival in the soil seed bank of Stipa bungeana, a grass species used in restoration of degraded land on the Loess Plateau in northwest China. Dormancy of fresh seeds was determined by incubation of seeds over a range of temperatures in both light and dark. Seed germination was evaluated after mechanical removal of palea and lemma (hulls), chemical scarification and dry storage. Fresh and one-year-stored seeds were sown in the field, and seedling emergence was monitored weekly for 8 weeks. Furthermore, seeds were buried at different soil depths, and then retrieved every 1 or 2 months to determine seed dormancy and seed viability in the laboratory. Fresh seeds (caryopses enclosed by palea and lemma) had non-deep physiological dormancy. Removal of palea and lemma, chemical scarification, dry storage (afterripening), gibberellin (GA₃) and potassium nitrate (KNO₃) significantly improved germination. Dormancy was completely released by removal of the hulls, but seeds on which hulls were put back to their original position germinated to only 46%. Pretreatment of seeds with a 30% NaOH solution for 60 min increased germination from 25% to 82%. Speed of seedling emergence from fresh seeds was significantly lower than that of seeds stored for 1 year. However, final percentage of seedling emergence did not differ significantly for seeds sown at depths of 0 and 1 cm. Most fresh seeds of S. bungeana buried in the field in early July either had germinated or lost viability by September. All seeds buried at a depth of 5 cm had lost viability after 5 months, whereas 12% and 4% seeds of those sown on the soil surface were viable after 5 and 12 months, respectively.     

Phylogenetic analysis on the bacteria producing non-volatile fungistatic substances

This study characterized the soil bacteria producing non-volatile fungistatic substances. Among the 2,100 colonies of soil bacteria randomly isolated from seven agricultural soil samples, 518 isolates (24.67% of total) showed fungistatic activity toward nematophagous fungi Paecilomyces lilacinus and Trichoderma viride by producing non-volatile substances. A phylogenetic analysis based on amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequence placed the 518 bacteria in three groups of the domain Bacteria: Actinomycetales, Bacillales, and Gammaproteobacteria. Three genera, Arthrobacter, Bacillus, and Pseudomonas, were the most frequently encountered groups.     

Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.)

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.     

木霉几丁质酶基因克隆及植物转化载体构建

利用PCR技术从绿色木霉LTR-2(Trichoderma virede)基因组DNA中扩增到一段序列,测序结果表明,该编码基因片段大小为 1 508 bp,其中包括一个 1 459 bp的开放阅读框,起始密码子位于 45 bp,终止密码子位于 1 501 bp,共编码氨基酸 424 个.在Genbank中进行序列比对,发现该序列同已发表的Trichoderma viride 42 ku几丁质酶氨基酸序列具有99%的同源性.将该片段同pCAMBIA1300中的35S启动子和35S-polyA终止子连接后,插入载体pCAMBIA1302多克隆位点中,构建成植物转化载体,最后将构建好的载体pCHI1302-42通过转化导入根癌农杆菌LBA4404中,为进一步构建转基因植物奠定了基础.     

The effect of azalomycin F on Ca2+ homeostasis in Trichoderma viride and Saccharomyces cerevisiae

Azalomycin F (AMF), a macrocyclic lactone antibiotic, in concentrations of 10(-5) g/ml (10(-6) - 10(-5) mol/l) was found to stimulate both the 45Ca2+ influx and efflux in intact Trichoderma viride submerged mycelium and in cells of Saccharomyces cerevisiae without having Ca2+ ionophoric properties. AMF also inhibited ATP-dependent Ca2+ uptake in membrane fractions prepared from T. viride submerged mycelium. 45Ca2+ which had been accumulated in membrane fractions in an ATP-dependent manner was released upon addition of AMF. This release was observed in light organellar fractions (LOF) of S. cerevisiae and of T. viride submerged mycelium and, to a small extent, in heavy organellar fraction (HOF) of S. cerevisiae. No Ca2+ releasing effect of AMF was observed in HOF from T. viride submerged mycelium. In S. cerevisiae expressing Ca2+-dependent photoprotein aequorin, AMF induced transients of luminescence which reflect changes in the cytoplasmic Ca2+ concentration. The results suggest that the stimulation by AMF of the Ca2+ efflux from the mycelium (cells) could be explained by an increase of the cytoplasmic Ca2+ concentration due to the release of Ca2+ from microsomal membranes or to the stimulation of Ca2+ influx.