An approach toward optimization of the influential growth determinants of opportunistic yeast isolate Pichia guilliermondii

The present study reports statistical optimization of growth conditions of an opportunistic fungal strain Pichia guilliermondii, isolated from the blood of patients suffering from bancroftian filariasis. Seven key determinants, namely, primary inoculums size (%), volume (mL) and pH of media, serum proportion, temperature (°C), incubation time (hr), and agitation speed (rpm) that influence in vitro growth of the pathogen were optimized statistically using response surface methodology (RSM). RSM with seven factors and two-level Box–Behnken design was employed for designing experimental run, prediction of case statistics, suitable exploration of quadratic response surfaces, and constructing a second-order polynomial equation. Analysis of variance (ANOVA) showed that primary inoculums size, volume of culture media, temperature, incubation time, and agitation speed exert most significant influence over fungal growth. The RSM study predicted that optimum fungal growth can be obtained using 10% primary inoculums size in 100 mL culture media with pH 6.0, 6.28% serum, 32.5°C temperature, and 24 hr of incubation, alongside agitation speed at 400 rpm. The desirability of the optimized growth model for P. guilliermondii is 99.123%, which indicated its accuracy and acceptability. Finally, the optimized growth module illustrated in the study could be useful in improving in vitro growth of clinically important P. guilliermondii.     

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO‐111 against blister blight disease of tea

Aims

The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition.

Methods and Results

A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO‐075, BMO‐111 and BMO‐147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36‐h‐old culture of BMO‐111 (1 × 10⁸ colony‐forming units ml^?1) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO‐111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO‐111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses.

Conclusions

It could be concluded that the biocontrol agent O. anthropi BMO‐111 was effective against blister blight disease of tea.

Significance and Impact of the Study

Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.     

Effects of different factors on the production of cellulase byCurvularia lunata

A purified culture of the fungal pathogenCurvularia lunata causing leaf blight of pearl millet was used for studies on the production of cell-wall-degrading enzymes. Czapek-Dox medium was found to be the best medium of the five different nutrient media used for the production of cellulase and growth of the fungus. Pectolytic enzymes could not be detected under different cultural conditions. Two-week incubation period, pH 6.0 and temperature 25°C were found to be the most favorable conditions for good growth and maximum production of cellulase by the fungus in present studies. The results are presented and discussed in the light of the earlier findings onC. lunata.     

Characterization of an antifungal soil bacterium and its antagonistic activities against Fusarium species

Bacteria were isolated from a cultivated soil and screened for antagonistic activity against Fusarium graminearum, a predominant agent of ear rot and head blight in cereal crops. Based on its in vitro effectiveness, isolate D1/2 was selected for characterization and identified as a strain of Bacillus subtilis by phenotypic tests and comparative analysis of its 16S ribosomal RNA gene (rDNA) sequence. It inhibited the mycelial growth of a collection of common fungal phytopathogens, including eight Fusarium species, three other ascomycetes, and one basidiomycete. The cell-free culture filtrate of D1/2 at different dilutions was active against macroconidium germination and hyphal growth of F. graminearum, depending on the initial macroconidium density. It induced the formation of swollen hyphal cells in liquid cultures of this fungus grown from macroconidia. A bioassay also demonstrated that D1/2 offered in planta protection against the damping-off disease in alfalfa seedlings caused by F. graminearum, while the type strain of B. subtilis was ineffective. Hence, B. subtilis D1/2 or its culture filtrate has potential application in controlling plant diseases caused by Fusarium.     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.     

Efficacy ofTrichoderma harzianum (Rifaii) on inhibition of ascochyta blight disease of chickpea

Trichoderma harzianum is a widely distributed soil fungus that antagonies numerous fungal phytopathogens. In this study, interactions between theT. harzianum isolates andAscochyta rabiei in experiments on agar growth medium were studied. All testedT. harzianum isolates produced metabolite that inhibited growth ofA. rabiei the agent of ascochyta blight disease of chickpea in culture. Isolates ofT. harzianum produced chitinase and β-1,3-glucanase when grown in liquid cultures containingA. rabiel cell wall, laminarin and chitin as sole carbon sources. Levels higher of these enzymes were induced inT. harzianum T15 isolate.     

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

Lactic acid bacteria in the inhibition of Fusarium graminearum and deoxynivalenol detoxification

Aims: Considering the agronomic and industrial damage that is caused by the fungus Fusarium graminearum, as well as the serious health risks it poses to humans and animals exposed to F. graminearum‐produced mycotoxin deoxynivalenol (DON), this study evaluated the ability of different lactic acid bacteria (LAB) strains to inhibit fungal development and remove DON in vitro. Methods and Results: The antagonistic effects of strains and commercial cultures of LAB were evaluated against F. graminearum IAPAR 2218 by the agar diffusion method. Additionally, the influence of the culture media, pH and the presence of lactic and acetic acid on these effects was tested. The capacity to remove DON by viable cells and heat‐inactivated cells was analysed in liquid media and quantified by high performance liquid chromatography (HPLC). All isolated strains and commercial cultures inhibited the fungus and removed DON. The pH and culture media concentration did not influence these abilities, but heat inactivation had a strong effect on the ability of bacteria to remove mycotoxin. Conclusions: The isolated bacteria are able to inhibit F. graminearum growth and remove DON in vitro. Significance and Impact of the Study: This study suggests potential application of the isolated LAB strains in the inhibition of F. graminearum IAPAR 2218 and DON removal in vitro.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor

Norway spruce ( Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μ m ) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.     

Bioprospecting Chemical Diversity and Bioactivity in a Marine Derived Aspergillus terreus

A comparative metabolomic study of a marine derived fungus (Aspergillus terreus) grown under various culture conditions is presented. The fungus was grown in eleven different culture conditions using solid agar, broth cultures, or grain based media (OSMAC). Multivariate analysis of LC/MS data from the organic extracts revealed drastic differences in the metabolic profiles and guided our subsequent isolation efforts. The compound 7‐desmethylcitreoviridin was isolated and identified, and is fully described for the first time. In addition, 16 known fungal metabolites were also isolated and identified. All compounds were elucidated by detailed spectroscopic analysis and tested for antibacterial activities against five human pathogens and tested for cytotoxicity. This study demonstrates that LC/MS based multivariate analysis provides a simple yet powerful tool to analyze the metabolome of a single fungal strain grown under various conditions. This approach allows environmentally‐induced changes in metabolite expression to be rapidly visualized, and uses these differences to guide the discovery of new bioactive molecules.     

Microplate Assay for Colletotrichum Spore Production

A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs.     

利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究

在胡椒（Piper nigrum Linn．)茎尖丛生增殖技术的基础上,以印尼大叶种“Lampong Type”无菌实生苗作外植体源,利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择。辣椒疫霉培养滤液的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著,过滤灭菌方式可以保持辣椒疫霉培养滤液的毒性,而高温高压灭菌方式则不能。随着辣椒疫霉培养滤液浓度的增加,茎尖和丛生芽的存活率和增殖率都在下降。在存活的茎尖或丛生芽培养中,一部分可正常增殖,其余的形成愈伤组织,或者保持生长停滞的休眠状态。在选择性培养基上继代培养2次后进行生根和移栽,利用离体叶片针刺接种法对温室条件下生长的移栽植株进行抗瘟病测定。以3次抗病检测均无明显症状的植株作为抗病株。随着辣椒疫霉培养滤液浓度的增加,得到的再生植株数量降低,但其中抗病株的比例提高。利用过滤灭菌方式加入选择性培养基的处理中,25％、50％和75％的辣椒疫霉培养滤液分别获得1株、4株和3株抗病株,分别占各处理再生植株总数的1．54％、20．00％和42．86％,共获得8株,占该组处理再生植株总数的8．70％。     

Changes in Saccharide Metabolism Induced by Infection of Camellia Sinensis by Exobasidium Vexans

Changes in saccharide contents of tea leaves during infection with blister blight fungus Exobasidium vexans Masse was studied. Saccharose and glucose contents decreased in the blistered portions when compared to the normal regions until sporulation and remained constant during the entire period of sporulation. Fructose content increased abruptly during the initiation of sporulation and remained constant up to the end of sporulation in both blistered and non-blistered regions. Starch content continuously decreased in the blistered region. Peroxidase activity was highly enhanced during the final stages of leaf senescence. The activity of acid invertase was inversely proportional to the starch content and closely related to the changes in the saccharose and glucose contents. Protein and chlorophyll contents gradually decreased in the blistered regions. This revised version was published online in July 2006 with corrections to the Cover Date.     

A survey of temperature and pH effect on colonial growth of Botryodiplodia theobromae RC1

Study of fungal colonial growth is a basic method to examine their behaviour in different cultivation conditions. The influence of temperature and initial pH on growth radial velocity and growth density of Botryodiplodia theobromae RC1, was studied in order to show the growth characteristics of this fungus. Both temperature and culture medium influenced growth density, but radial velocity of growth was only affected by temperatures above 40 degrees C. In addition, initial pH of culture media did not affect either parameter.     

Acid phosphatase distribution and localization in the fungus Humicola lutea

Acid phosphatase activities in a culture liquid and mycelial extract were studied in submerged cultures of the filamentous fungus Humicola lutea 120-5 in casein-containingmedia with and without inorganic phosphate (Pi). The Pi-repressible influence on the phosphatase formation was demonstrated. Significant changes in the distribution of acid phosphatase between the mycelial extract and culture liquid were observed at the transition of the strain from exponential to stationary phase. Some differences in the cytochemical localization of phosphatase in dependence of Pi in the media and the role of the enzyme in the release of available phosphorus from the phosphoprotein casein for fungal growth were discussed.     

Patterns of interaction between Populus Esch5 and Piriformospora indica: a transition from mutualism to antagonism

Piriformospora indica (Sebacinaceae, Basidiomycota) is an axenically cultivable, plant growth promoting root endophyte with a wide host range, including Populus. Rooting of Populus Esch5 explants started within 6 days after transfer to WPM medium. If such plantlets with roots were inoculated with P. indica, there was an increase in root biomass, and the number of 2nd order roots was increased significantly. A totally different observation was recorded when the explants were placed into WPM with pre-grown P. indica. The interaction led to complete blocking of root production and severely inhibited plant growth. Additionally, branched aerial roots appeared which did not penetrate the medium. On contact with the fungal colony or the medium, the ends of the aerial roots became inflated. Prolonged incubation stimulated the fungus to colonize aerial parts of the plant (stem and leaves). Mycelium not only spread on the surface of the aerial parts, but also invaded the cortical tissues inter- and intracellularly. Detached Populus leaves remained vital for 4 - 5 weeks on sterile agar media or on AspM medium with pre-grown P. indica. When the fungus was pre-grown on culture media such as WPM, containing ammonium as the main source of nitrogen, leaves in contact with the cultures turned brownish within 4 - 12 h. Thereafter, the leaves bleached, and about one day later had become whitish. Thus, cultural conditions could alter the behaviour of the fungus drastically: the outcome of the interaction between plant and fungus can be directed from mutualistic to antagonistic, characterized by fungal toxin formation and extension of the colonization to Populus shoots.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.