A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

Cultivation of Entomopathogenic Fungi for the Search of Antibacterial Compounds

Entomopathogenic fungi are a rich source of natural bioactive compounds. To establish cultivation conditions which facilitate the production of bioactive compounds and to select good genera among entomopathogenic fungi as the producer, 47 typical entomopathogenic fungi were tested for their ability to produce antibiotic activity. Thirty-eight strains (81%) and 30 strains (64%) of these fungi produced either anti-Bacillus compounds or anti-Staphylococcus compounds, respectively, indicating that the majority of the entomopathogenic fungi tested possessed the ability to produce antibacterial compounds. Using 9 representative strains (Aschersonia sp. HF724, Beauveria bassiana HF338, Cordyceps ramosopulvinata HF746, Metarhizium anisopliae HF293, Metarhizium flavoviride HF698, Nomuraea rileyi HF588, Paecilomyces fumosoroseus HF254, Paecilomyces tenuipes HF419, and Verticillium lecanii HF238), the cultivation conditions in liquid medium were surveyed with respect to the cultivation procedure and medium composition, particularly in terms of the presence or absence of insect-derived materials. At 26 °C, M. anisopliae HF293, N. rileyi HF588, and V. lecanii HF238 strains produced clear antibiotic activity against Bacillus and Saccharomyces, but only in the presence of insect-derived materials, suggesting that the production of antibacterial/antifungal compounds by entomopathogenic fungi is triggered by the presence of insect-derived materials.     

Effects of the fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an important citrus pest primarily because it transmits bacteria putatively responsible for huanglongbing, a serious disease of citrus. We present a study on the effects of blastospore and conidial formulations of Isaria fumosorosea Wize on feeding rates and mortality of adult psyllids in laboratory bioassays. Information on quantities of honeydew droplets was used to make inferences on feeding rates. Psyllids treated with the blastospore formulation of I. fumosorosea produced fewer honeydew droplets compared to the conidial treatment and control beginning within the first 24 h after treatment. The highest daily mean number of droplets thereafter never exceeded 2.4 drops compared to 4 and 8 for the conidial treatment and control, respectively. The mean number (±SEM) of honeydew droplets produced per psyllid per day over 7 days was significantly higher in the control (5.5±0.5) compared to the blastospore treatment; however, there were no significant differences between the treatments. Psyllids treated with the conidial formulation of the pathogen showed no significant reduction in feeding activity until 4 days after treatment. One and 2 day's post-exposure, mortality of psyllids in the blastospore treatment ranged from 8 to 25% compared to 0% in the conidial and control treatments. By 7 days post-exposure, psyllid mortality reached 100% under both fungal treatments compared to none in the controls. This study documented that adult psyllids infected by I. fumosorosea (PFR 97) produce less honeydew than healthy psyllids and suggests that they may feed less, which could potentially reduce the spread of huanglongbing.     

Production of red pigments by the insect pathogenic fungus Cordyceps unilateralis BCC 1869

Production of red pigments (naphthoquinones) by the insect pathogenic fungus Cordyceps unilateralis BCC 1869 was investigated in this study. Cultivation conditions, including temperature, intitial pH of medium, and aeration, were optimised to improve the yield of total naphthoquinones in shake-flask culture of C. unilateralis. The highest yield of total naphthoquinones (3 g L^–1) was obtained from a 28-day culture grown in potato dextrose broth with an initial pH of 7.0, at 28°C with shaking-induced aeration at 200 rpm. An extraction process for isolation of the targeted naphthoquinone, 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-1,4-naphthoquinone (3,5,8-TMON), from a culture of C. unilateralis, was also developed. The yield of 3,5,8-TMON obtained was about 1.2 g L^–1 or 40% of total naphthoquinones. The stability of 3,5,8-TMON was very high, even upon exposure to strong sunlight (70,000 lx), high temperature up to 200°C, and acid and alkali solutions at concentrations of 0.1 M     

Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota:Clavicipitaceae) and its evolutionary implications

Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts,traditionally included in the family Clavicipitaceae of Ascomycota.Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co-evolution of parasites and hosts in entomopathogenic ascomycetes.To this end,we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses.Based on the phylogenetic tree,we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character.The phylogenetic analyses revealed two important points.(i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps.The vertically immersed perithecia form was the ancestral state,superficial and obliquely immersed perithecia were derived characters,obliquely immersed was irreversible.Species with obliquely immersed perithecia were in a closely related group and were the derived group.(ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats.Based on the results of these analyses,host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

科码嘉培养基在病原微生物分离中的应用

为了快速、准确检测各种致病菌,为疾病诊疗赢得时间,有效地控制疾病和疫情的发生、发展,利用传统分离培养基和显色培养基同时检测样品,得出检测样品阳性率。科码嘉显色培养基比传统分离培养基省时、省力,阳性率高。科码嘉显色基制备简单、使用方便,缩短检验周期,提高了检测率。     

霉克舒对致病性浅部真菌的体外抑菌作用研究

从湛江地区 15 4例癣病患者分离真菌 ,评价一种新的抗真菌药—霉克舒对上述真菌菌株的体外抑菌活性。应用琼脂稀释法测定霉克舒对浅部真菌的最小抑菌浓度 (MIC) ,同时以兰美抒作为对照药物。从 15 4例癣病患者中分离出 14 1株真菌 ,其中以红色毛癣菌和须癣毛癣菌为主 ,分别占 6 6 .7%和 14 .2 %。霉克舒的抑菌作用与兰美抒相比略强或相当 ;对霉克舒各单一成分的初步抑菌效果进行比较 ,复合物的抑菌作用明显强于水杨酸或特比萘芬等单一成分。上述结果显示 ,霉克舒在体外对常见致病性浅部真菌具有较强的抗菌活性。     

Reassessment of type specimens of Cordyceps and its allies described by Dr. Yosio Kobayasi, preserved in the National Museum of Nature and Science. Part 2: Cordyceps (Elaphocordyceps) on Elaphomyces

Four holotype specimens of Cordyceps parasitic on Elaphomyces that were originally described by Dr. Yosio Kobayasi and Mr. Daisuke Shimizu were relocated, and TNS (National Museum of Nature and Science, Japan) registration numbers (TNS-F-number) were assigned: viz. Cordyceps minazukiensis (TNS-F-197989), C. ophioglossoides var. cuboides (TNS-F-230312), C. valvatistipitata (TNS-F-230284), and Cordyceps × jezoensoides (TNS-F-230286). Two specimens were selected as lectotypes: C. delicatistipitata (TNS-F-230293) and C. ophioglossoides f. alba (TNS-F-18223). Those taxa, except for Cordyceps × jezoensoides, have been treated as members of the genus Elaphocordyceps in the latest taxonomy. The new combination Elaphocordyceps × jezoënsoides (Kobayasi) is proposed. In addition, Elaphomyces asahimontanus Kobayasi, the host of Cordyceps delicatistipitata, is lectotypified (TNS-F-230293).     

孝顺竹愈伤组织增殖培养基优化研究

为筛选适宜孝顺竹愈伤组织继代增殖培养基,控制褐变发生,提高再生体系效率,对培养基组成如5种基本培养基、6种有机添加物、7种糖类和5种大量元素等因子进行试验分析。结果表明：培养20 d,基本培养基以MS效果较好,愈伤组织增殖2.8倍,白至淡黄色,致密;有机添加物以1.0 g·L^-1脯氨酸效果较显著,愈伤组织增殖3.64倍,淡黄色,致密均一;碳源以30 g·L^-1麦芽糖效果较好,愈伤组织增殖2.96倍,白至淡黄色,致密。5种大量元素中NH₄NO₃对孝顺竹愈伤组织增殖的影响达到显著水平,以825 mg·L^-1为较佳浓度,培养29 d愈伤组织增殖可达5倍以上,部分出现根分化;适宜孝顺竹愈伤组织培养的大量元素组合为：KNO₃ 475 mg·L^-1+NH₄NO₃ 825 mg·L^-1+MgSO₄·7H₂O 185 mg·L^-1+KH₂PO₄ 340 mg·L^-1+CaCl₂·7H₂O 440 mg·L^-1。     

Selection of fungi for the control of Musca domestica in aviaries

Musca domestica is a major problem in poultry production where management has relied mostly on pesticides, which eventually have led to the development of widespread resistance in insect populations and environmental contamination. In this study, we explored the use of entomopathogenic fungi for the control of houseflies in Brazil. A total of 10 isolates of different fungal pathogens were tested on the larval stages of M. domestica, which were originally derived from a commercial aviary. Among the test fungi, Metarhizium anisopliae (sensu stricto) CG46 and CG30 were the most virulent isolates, causing up to 60% mortality at day 10, but only CG46 was selected for a field trial as it attained higher sporulation on larval cadavers. A commercial product based on the M. anisopliae isolate ESALQ-1037 (Metarril®) was also field-tested using standard treatment procedures. After spraying both fungi in the aviary manure, there was a significant reduction (twofold lower) in adult density of M. domestica captured per trap compared to the control shed. Therefore, our results indicate that M. anisopliae is a promising biological control agent for M. domestica larvae, but new strategies for fungal application and appropriate formulation need to be developed to improve its effectiveness in poultry environments.     

The potential of Isaria spp. as a bioinsecticide for the biological control of Nasutitermes corniger

The termite Nasutitermes corniger is a serious pest infesting urban areas of Brazil and many other countries. Control largely depends on synthetic pesticides whose indiscriminate use can impact the environment and the health of humans and other animals. Alternative strategies against insect pests, such as biological control by entomopathogenic fungi, could be effective while minimising these deleterious effects. We analysed the actions of the entomopathogenic fungi Isaria farinosa, Isaria fumosorosea, and Isaria javanica against the insect N. corniger. Our results indicated that the fungi examined were pathogenic against N. corniger, with I. farinosa ESALQ1355 being the most efficacious strain, resulting in the death of 95% of the workers (LC₅₀ 6.66?×?10⁴ conidia/mL) and 85% of the soldiers (LC₅₀ 6.81?×?10⁴ conidia/mL). This is the first report of the pathogenicity of Isaria spp. on N. corniger. These in vitro results suggest that I. farinosa ESALQ1355 demonstrates a significant biological potential for controlling N. corniger.     

高产蛹虫草菌株发酵培养基的研究

在单因素试验初步确定高产蛹虫草菌株发酵培养基的基础上,以蛹虫草茵丝体中腺苷含量为指标,进行11因素2水平Plackett—Burman试验设计试验,结合多元一次回归模型和F检验方法,筛选出发酵培养基中影响显著的组分酵母浸粉、蔗糖和维生素B1,采用旋转中心组合设计方法对这三个组分进行进一步优化,结合多元二次回归模型和响应面分析,获得高产蛹虫草菌株的最佳培养基（g／L）：蔗糖18．85、蛋白胨10、酵母浸粉18．97、KH2PO,3、MgSO4 3、维生素Bl0．235、ZnCl20．011、（NH4）2S0410。验证试验结果表明蛹虫草腺苷得率较单因素优化获得的发酵培养基提高了26．91％。     

The Cultivation of Symbiote-free Marine Ciliates in Axenic Medium

SYNOPSIS. Media have been developed for axenic cultivation of 10 strains belonging to 7 species of small marine ciliates. A medium containing cerophyl extract, proteose peptone, trypticase, yeast nucleic acid, biotin, calcium pantothenate folic acid, nicotinamide, pyridoxal HCl, riboflavin, thiamine HCl, and DL-thioctic acid in sea water supports the growth of Uronema nigricans strains Pc and 34/2, Parauronema virginiatum strains 2/1 and 19/1, Miamiensis avidus strains Ma and 19/3, Miamiensis sp. strain 1/1, a strain of "G" ciliate, and strains 33/8 and 34/7 of 2 unidentified species. By substituting a mixture of asolecithin, cephalin, and Tween 80 for cerophyl in the medium, luxurious growth of all except the strains of the 2 unidentified species can be obtained. A defined medium consisting of 18 amino acids, 5 purine derivatives, 8 vitamins, asolecithin, cephalin, and Tween 80 in synthetic sea water also has been developed for 6 of the strains: M. avidus Ma and 19/3, Miamiensis sp. 1/1, P. virginiatum 2/1 and 19/1, and U. nigricans Pc. In general the ciliates grow best at pH 7.2 in the dark at 27 C in media containing sea water of density = 1.015. Under these conditions maximum populations are reached in 4–5 days and range from several hundred thousand to 3 or 4 × 10⁶ depending upon the strain. Electronmicroscopic observations for the presence of endosymbiotes gave negative results.     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

酵母产γ-氨基丁酸发酵培养基的优化

目的：提高酵母产γ-氨基丁酸的能力。方法：采用单因素及正交设计实验对酵母产γ-氨基丁酸（GABA）的培养基进行优化。结果：确定最适碳源为葡萄糖,最佳氮源为蛋白胨和硫酸铵复合氮源,合适的无机盐为KH2PO4;最佳发酵培养基为3%葡萄糖,3%蛋白胨,0.3%（NH4）2SO4和0.1%KH2PO4。在此培养条件下,摇瓶发酵可以获得1.690g.L-1的GABA产量。结论：发酵培养基的优化,提高了菌株产γ-氨基丁酸的能力。     

Selection of an Isolate of the Insect Pathogenic Fungus Metarhizium anisopliae Virulent to the Lettuce Root Aphid, Pemphigus bursarius

The virulence of hyphomycete entomopathogenic fungi was measured in laboratory bioassays against the lettuce root aphid, Pemphigus bursarius, a serious pest of field lettuce grown in the UK. Of 25 isolates of fungi examined, only one isolate, Metarhizium anisopliae 391.93, killed lettuce root aphids consistently. This fungus was isolated originally from the closely related saltmarsh aphid, P. trehernei. The median lethal concentration of conidia at 10 days post6- 1 inoculation estimated from five independent bioassays was 2.45 × 10⁶ conidia ml^-1. The fungus had no significant effect on the mean number of offspring/aphid produced, but it sporulated 6 profusely on host cadavers, producing approximately 4 × 10⁶ conidia/cadaver 14 days after treatment, and diseased aphids died attached to plant roots. It thus has the potential to spread through densely packed colonies of P. bursarius feeding on the roots of susceptible or partially resistant plants.     

Laboratory studies on the competition for insect haemocoel between Beauveria bassiana and Steinernema ichnusae recovered in the same ecological niche

Laboratory assays evaluated the combined action of the entomopathogenic nematode Steinernema ichnusae and the entomopathogenic fungus Beauveria bassiana isolated from the same ecological niche, an oak forest in Sardinia (Italy). Galleria mellonella larvae were used as the test insect with the aim of understanding what happens in host haemocoel during a simultaneous infection with two different entomopathogens. Larval mortality assays were performed using nematodes and fungi both alone and together, at the same and different times, and in different concentrations. No additive or synergistic effects were observed, but there was a clear antagonism and competition for survival space in the haemocoel. Moreover, mutual effects between the symbiotic bacteria of the entomopathogenic nematode Xenorhabdus bovienii and entomopathogenic fungi were investigated. In laboratory experiments, X. bovienii crude extracts were tested for their activity against fungal growth. Compounds produced by B. bassiana were tested for their activity against the growth of bacteria, revealing that X. bovienii and B. bassiana are antagonistic to each other.     

应用响应面法优化嗜热脂肪土芽孢杆菌培养基

目的：筛选并优化嗜热脂肪芽孢杆菌CHB1液体发酵培养基。方法：通过Plackett-Burman试验和响应面分析方法,确定培养基的主要影响因素和最佳浓度。结果：利用SAS软件进行分析,确定对响应值影响最大的3个因素为豆粕、酵母粉、K2HPO4。最佳培养基组成为0.51%、豆粕浓度为0.425%、K2HPO4浓度为0.994%。根据模型预测得到的理论最大菌数为2.94×10^8cfu。在初始条件下实验,菌数为2.40×10^8cfu,在优化的最佳培养基条件下,实际的菌数为3.06×10^8cfu。菌数比优化前提高了27.3%。试验值与预测值的误差为4.08%。结论：实验值与模型预测值拟合良好。