Liquid culturing of microsclerotia of Mycoleptodiscus terrestris,a potential biological control agent for the management of hydrilla

Mycoleptodiscus terrestris has potential as an inundative biological control agent for the management of hydrilla, one of the world’s worst aquatic weeds. Essential to producing a marketable bioherbicidal product was the development of liquid culture procedures that would yield propagules that maintained biocontrol efficacy. Since M. terrestris did not produce conidia in liquid culture, various nutritional conditions were evaluated as a means to produce high concentrations of stable fungal propagules such as microsclerotia. Evaluations of propagule formation and biomass yield were carried out in liquid culture media containing a basal salts solution amended with corn steep liquor powder or cottonseed meal combined with 4% or 6% glucose. Hyphal aggregation was observed by day 2, and by day 8 abundant melanized microsclerotia were present in the broth cultures. When applied as a liquid inoculum to hydrilla at rates of 0.1 and 0.2 ml/l, the microsclerotial matrix was capable of significantly reducing hydrilla shoot biomass by as much as 99%. Air-dried microsclerotia were capable of hyphal germination in 24 h and sporogenic germination in 72 h. These capabilities have significance for the use of microsclerotia of M. terrestris as the preferred inoculum for biocontrol purposes. Hyphae germinating from microsclerotia on hydrilla plant surfaces can establish initial infection sites followed several days later by secondary infections resulting from the development and release of spores from the surface of the microsclerotia. The capability of microsclerotia of M. terrestris to remain stable as a dry preparation and to germinate both hyphally and sporogenically upon rehydration enhances the potential of this fungus for use as a nonchemical, biological control agent for hydrilla.     

Factors affecting spore germination in Aspergillus niger

Temperature markedly affected germination and germ tube length of A. niger. More than 90% of the spores were germinated in the range 30°–34 °C and formed maximum length of germ tubes. At temperatures from 38° to 43 °C, the proportion of the spores that germinated as well as the germ tube length were both gradually decreased. However, at 47°C germ tube formation was completely inhibited up to 15 hrs. after inoculation.High relative humidity was found necessary for the spore germination of A. niger. Germination failed to occur at 76% relative humidity. At 78 and 81% relative humidity germination was detected 15 hrs. after inoculation while at the higher humidities germination was started after 6 hrs. only.Conidiospores of A. niger were very sensitive to changes in the hydrogen ion concentration, pH. Complete inhibition of germination was found at pH less than 3.5. The germination and the length of the formed germ tubes increased with pH to reach their maximum rates at pH 4.5.     

Identification of VdASP F2-interacting protein as a regulator of microsclerotial formation in Verticillium dahliae

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species. The melanized microsclerotia enable V. dahliae to survive for years in soil and are crucial for its disease cycle. In a previous study, we characterized the secretory protein VdASP F2 from V. dahliae and found that VdASP F2 deletion significantly affected the formation of microsclerotia under adverse environmental conditions. In this study, we clarified that VdASP F2 is localized to the cell wall. However, the underlying mechanism of VdASP F2 in microsclerotial formation remains unclear. Transmembrane ion channel protein VdTRP was identified as a candidate protein that interacts with VdASP F2 using pull-down assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and interaction of VdASP F2 and VdTRP was confirmed by bimolecular fluorescence complementary and coimmunoprecipitation assays. The deletion mutant was analysed to reveal that VdTRP is required for microsclerotial production, but it is not essential for stress resistance, carbon utilization and pathogenicity of V. dahliae. RNA-seq revealed some differentially expressed genes related to melanin synthesis and microsclerotial formation were significantly downregulated in the VdTRP deletion mutants. Taken together, these results indicate that VdASP F2 regulates the formation of melanized microsclerotia by interacting with VdTRP.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

Factors Affecting Spore Germination of Volvariella volvacea

Environmental factors affecting basidiospore germination were studied with Volvariella volvacea, the edible straw mushroom which is common in South East Asia. A relatively mild heal shock is necessary for spore germination. The spores give best germination at 40°C although early hyphal growth is better at 35°C the germination of spores is affected by temperature. pH. a presoaking treatment and spore density. Higher pH supports more germination but seems unfavourable for early mycelial growth. Presoaking treatment in phosphate buffer solution or distilled water also stimulates germination markedly.     

Certain aspects of the pH dependence of triggering inBacillus megaterium spores

Incubation of unactivatedBacillus megaterium 14581 spores in glucose, or in glucose plusl-alanine, at or below pH 3.6 resulted in germination arrested somewhere before onset of stainability. However, triggering continued at this reduced pH, and spores thus triggered were fully capable of completing the germination sequence in the absence of the germinants once the pH was neutralized. The same spores could be triggered either by a mixture of glucose andl-alanine or by a larger concentration of glucose alone. From this it was concluded that triggering results from an adequate stimulus which can be generated in different ways.l-alanine action in triggering has a pH profile distinct from that of glucose, suggesting that these two germinants have different receptor sites as well. At a level of acidity at which a weak glucose concentration triggered relatively few spores, a much larger fraction was found apparently distributed over a range of sub-triggering levels. Some of these could be made to trigger on transfer to a secondary reagent, or mixture of reagents, which by themselves are not very efficient germinants of the strain studied. The degree of additional triggering was found to depend on the nature of the complementary germinants, as well as on the pH at which glucose stimulated them. Evidence that spores may occupy stimulated states for finite lifetimes is presented and discussed.     

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.     

根系土壤假丝酵母菌Candida sp. YIN9对大丽轮枝菌和全齿复活线虫的抑制作用

[目的] 研究黄萎病抗性棉(海 7124)根际土壤中酵母菌株对棉花黄萎病病原真菌大丽轮枝菌和全齿复活线虫的拮抗效果,为生物防治棉花黄萎病和全齿复活线虫提供理论依据。[方法] 通过镜检、糖发酵实验、碳源同化实验、26S rRNA测序对菌株的形态、生理生化特征及其系统发育关系进行鉴定,并利用七叶苷筛选、刚果红染色、平皿对峙实验、盆栽实验、平板生测实验测试其产酶活性以及抑制大丽轮枝菌和杀线虫活性。[结果] 从大批黄萎病抗性棉(海 7124)根际土壤中筛选出编号为YIN9的酵母菌菌株,分类鉴定结果表明:YIN9菌株属于假丝酵母属Candida。平皿对峙实验结果表明:菌株YIN9对大丽轮枝菌的抑菌率达59%;将菌株YIN9的无菌发酵滤液与大丽轮枝菌孢子共培养12 h后镜检发现,用菌株YIN9处理的实验组,大部分棉花黄萎病病菌孢子不能正常萌发。盆栽实验结果表明:菌株YIN9对棉花黄萎病的平均防治效果为60.02%,可以显著降低感病棉棉花黄萎病的发病率和病情指数。此外,与从黄萎病抗性棉根际土壤中筛选获得的其他酵母菌株相比,菌株YIN9具备较高的杀线虫活性:菌株作用全齿复活线虫48 和60 h后,线虫死亡率分别为90%和100%。将菌株YIN9发酵液煮沸后,其抑制大丽轮枝菌和杀线虫活性均急剧下降,进一步测试发现,该菌株拥有较高的蛋白酶和纤维素酶活性。[结论] YIN9中的生防因子可能是热不稳定性物质,具备较高的杀线虫活性,可以显著提高感病棉对黄萎病的抗性。     

The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.     

Effect on germination and post-germination development of Colletotrichum dematium var circinans due to light and dark incubation and coverslip placement

Light regime was not a major factor in germination of spores of Colletotrichum dematium var circinans. Number of germ-tubes and appressoria formed were affected by incubation regime and coverslip placement. Most germ-tubes and appressoria were produced on spores under shimmed coverslips. Light incubation favored germ-tube production and dark incubation favored appressoria production. Predicted time to 100% germination was affected by treatments. Shortest times were predicted for light incubated spores under shimmed coverslips. Longest times were predicted for non-coverslipped spores. A significant number of appressoria were produced sessile on spores. Most sessile appressoria were produced on dark incubated spores. Approximately 56 % of all spores germinated, and approximately 60% of all germ-tubes and appressoria were produced on these spores which were situated closest to coverslip or droplet edges. Placement of germ-tubes on spores was not affected by treatments. However, percentages of germ-tubes, initiated from various areas of spores, were significantly different. This variation appears to be due to factors internal to spores.     

FUNGISTASIS IN SOILS

1. Fungistasis in soil is a widespread phenomenon affecting most fungal propagules, though some are insensitive. In most instances, it is coexistent with the presence of living microorganisms, and is annulled by energy-yielding nutrients. Fungistasis with characteristics similar to that in soil may also occur on leaves of plants. 2. Germination and growth of bacteria and actinomycetes is also restricted in soils. The characteristics of their inhibition appear to be the same as those for fungi. Therefore, the concept of a widespread microbial inhibition in soil can be applied to all three groups of microorganisms. 3. Fungistasis can be detected by various direct methods, or indirectly by methods involving the use of porous or permeable carriers. It may be expressed as a restriction on the final amount of germination (the usual parameter), germination rate (with time), and rate of germ-tube or hyphal growth. Since the expression of fungistasis is often complete in soil, titration with nutrients may be required to distinguish between the sensitivities of different fungi. 4. Fungistasis generally is expressed most strongly at soil moisture contents somewhat less than saturation. Its expression usually is maximal in neutral or slightly alkaline soils. In acidic conditions fungistasis may be lessened because of suppression of bacterial and actinomycete activity. Increased sensitivity of some fungi in soils of pH > 7.0 may be caused by a directly unfavourable effect of pH on the fungus. 5. Fungal species with small spores tend to be highly sensitive to fungistasis. These spores tend to germinate slowly and to require exogenous nutrients for germination. By contrast, species with larger spores and sclerotia often do not require exogenous nutrients for germination. The larger spores tend to germinate rapidly and to exhibit low sensitivity, as compared with small spores. A few nutrient-independent spores are insensitive to fungistasis. At least a part of the difference in sensitivity is related to germination time; spores which germinate slowly compete poorly with the soil micro-flora for their nutrients. 6. Fungistasis is often temporarily annulled by enriching the soil with energy-yielding nutrients. Usually, complex materials such as plant residues are most effective. A few weeks after such treatment, the level of fungistasis may, however, be increased. Annulment of fungistasis by compounds not utilized as energy sources has not yet been demonstrated. 7. Several soils naturally suppressive to Fusarium wilt diseases were more fungistatic to Fusarium than soils conducive to wilt. Potential means by which fungistasis may be manipulated to control root-infecting fungi are (a) through stimulation of germination with nutrients, thus exposing the germ tube to lysis, and (b) by increasing the fungistatic level of soil through appropriate amendments. 8. Volatile substances identified in soils, some of which are potentially inhibitory to fungi include (a) ammonia, which apparently is evolved from ammonium salts in some arid soils of high pH, (b) ethylene, which has been identified in some soils of pH < 7.0 (though high levels of this gas seem to be tolerated by most fungi), (c) allyl alcohol, and (d) other unidentified substances. Non-volatile inhibitors include high molecular weight substances revealed by molecular sieve chromatography of soil extracts. Microbial metabolites such as those present in staled fungal cultures also have been proposed to account for fungistasis. In a few soils fungistasis persists after sterilization because of the presence of inhibitory concentrations of calcium carbonate, iron or aluminium. Inherent in the proposition that inhibitory substances provide the primary mechanism of fungistasis is the concept of a highly complex phenomenon, involving various highly specific inhibitory and counteracting stimulatory substances, with the outcome for the fungus depending on the kinds and relative amounts of each present. 9. By the nutrient-deficiency hypothesis, the level of available nutrients in soil is insufficient to support germination of nutrient-dependent propagules, except in nutrient-rich microsites. Inhibition of nutrient-independent propagules is explained by loss of endogenous nutrients required for germination, through microbial nutrient competition. Evidence for this hypothesis is (a) the imposition of fungistasis on numerous nutrient-independent propagules during incubation on leaching model systems designed to simulate microbial nutrient competition in soil, (b) similar losses of endogenous nutrients occurring on soil and the leaching system, and (c) the fact that soils are chronically deficient in energy in relation to the microbial populations present, with the consequence that enforced inactivity is imposed upon most of the population at any given time for this reason alone, regardless of the presence or absence of fungistatic substances. Journal series article no. 7747 from the Michigan Agricultural Experiment Station.     

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.     

黄萎病不同发生程度棉田中土壤微生物多样性

作物根际土壤微生物群落对土壤生态及作物健康至关重要。以棉花黄萎病不同发生程度棉田的土壤为研究对象,采用理化分析、微生物纯培养及高通量测序技术对土壤理化性质及微生物数量、细菌丰度多样性进行综合分析。结果表明:在纯培养条件下,大丽轮枝菌无菌发酵滤液对细菌生长有明显的抑制作用;棉田接种大丽轮枝菌对土壤中细菌、真菌、放线菌的数量及细菌菌群丰度多样性未产生明显影响,不同采样时间的土壤中细菌菌群结构差异更大。土壤中大丽轮枝菌微菌核数量与棉花黄萎病的发生程度呈显著正相关。土壤肥力对土壤中微生物数量起主导作用,而水稻-棉花轮作能够使棉田有效降盐、减病、改善土壤肥力。通过生物防治、作物轮作、深翻等调控措施增加土壤中有益菌群数量、改善土壤生态、降低棉田土壤中大丽轮枝菌菌源数量是减轻棉花黄萎病危害的基础。     

Bacterial growth on N,N-dimethylformamide: implications for the biotreatment of industrial wastewater

The degradation of N,N-dimethylformamide (DMF) by bacterial consortia was investigated under aerobic, fermentative and nitrate-reducing conditions and a variety of salt concentrations (0.2%, 4% and 7% NaCl w/v) and pH values (5 and 7). Optimization of degradation conditions was studied to provide information and recommendations for large-scale biological treatment processes. Under aerobic conditions, mineralization of DMF (200 mg l⁻¹, 2.7 mM) was achieved under all combinations of salinity and pH. The rate of bacterial growth decreased with increasing salinity. Changes in the salt concentration and pH still resulted in mineralization and unchanged yield of bacterial cells. At 0.2% NaCl and either pH 5 or 7, growth occurred on DMF in the range 0.2–1 g l⁻¹. However, cell yield decreased with increasing concentrations of DMF. Under conditions of 0.2% NaCl, pH 7 and 4% NaCl, pH 5, growth on DMF at 5 g l⁻¹ resulted in the production of an intermediate that was detected using gas chromatography (GC). It is proposed that the intermediate was dimethylamine, and its persistence in growth media was attributed to suppressed growth as a result of an increase in pH. A culture capable of degrading DMF under nitrate-reducing conditions was obtained at 0.2% NaCl and pH 7, but not at more saline and acidic conditions. Growth and degradation of DMF were considerably slower under these conditions compared with aerobic conditions. Fermentative degradation of DMF was not observed. Journal of Industrial Microbiology & Biotechnology (2000) 25, 8–16. Received 14 July 1999/ Accepted in revised form 30 March 2000     

Streptomyces plicatus as a model biocontrol agent

Three hundred and seventy two isolates belonging to the genusStreptomyces were isolated and screened for chitinase production.Streptomyces plicatus was found to be the best producer. The highest chitinase production were incubated for 3 d at 30 °C on buffered culture medium (pH 8.0) containing chitin plus sucrose and calcium nitrate as carbon and nitrogen sources.S. plicatus chitinase had a highly significant inhibitory effect on spore germination, germ tube elongation and radial growth ofFusarium oxysporum f.sp.lycopersict., Altrernaria alternata andVerticillium albo-atrum, the causal organisms ofFusarium wilt, stem canker andVerticillium wilt diseases of tomato. Application ofS. plicatus to the root system of tomato plants before transplantation markedly protected tomato plants against the tested phytopathogenic fungiin vivo.