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15 May 2012, Volume 31 Issue 3
    

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  • Mycosystema. 2012, 31(3): 305-306.
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    Many Chinese mycologists are involved in basic and applied studies of different entomopathogenic fungi and have been making great achievements. In this special issue on entomopathogenic fungi, one review and different research papers were published including the resource investigations, gene cloning and the analysis of bioactive metabolites of different entomopathogenic fungi. To improve the application efficacies of mycoinsecticides and medicinal insect fungi, further studies are still required to investigate the virulence gene functions and molecular interactions between fungi and insects etc.
  • Review
  • Mycosystema. 2012, 31(3): 307-321.
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    Scale insects (Hemiptera: Coccoidea) compose a group of important plant pests of agricultural crops, forest plants, ornamental plants and fruit trees. The history of the study of entomopathogenic fungi as a biological insecticide is reviewed according to the three developmental stages: the pioneering stage, the slow development stage, and the prosperity stage. Additionally, the status of this field in China was discussed. A list including approximately 140 species within 55 genera of the recorded fungal pathogens of scale insects in the world was provided. Finally, we provide four suggestions for the development of entomopathogenic fungi in the future.
  • Papers
  • Mycosystema. 2012, 31(3): 322-330.
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    A new fungal pathogen infecting larvae of Cnaphalocrocis medinalis was found during a survey of pathogens of the insect in Guangdong, China in 2006. The fungus was cultured in vitro and was identified as Zoophthora radicans. This is the first report of Z. radicans infecting larvae of C. medinalis in China. The effects of temperature and photoperiod on radial growth of the fungus were studied in the laboratory. The results showed that the optimum temperature for mycelial growth was 25℃ though it could grow at 15-25℃, and the photoperiods also affected insignificantly on mycelial growth. The fungus mainly infected the 3rd, 4th and 5th instar larvae and the infection rates were 2.71%, 24.32% and 72.97%, respectively. The epizootics of the fungus was consistently high during October-November, with infection rate reaching 95%. The pathogen could survive during unfavourable season in rice field habitat in the form of resting spores and initiated epizootic in the following year. Thus, the fungus acted as a successful natural suppression agent of the leaf folder population.
  • Mycosystema. 2012, 31(3): 331-340.
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    Nine strains of different species of Metarhizium, including strain of M. flavoviride (Mf82) newly isolated from Nilaparvata lugens (St?l), and eight M. anisopliae strains from lab collection, were tested against the adults of N. lugens. The chitinase activities of these strains were also assayed. The results showed that both the cumulative mortality (82.1%) and the chitinase activity (9.78U/mg) of strain Mf82 were the highest among all tested strains. Scanning electron microscope (SEM) observations showed the germinating conidia of the strain Mf82 penetrated the cuticle not only via the intersegmental membranes and folded regions, but also through the pronotum which contained a large quantity of chitin. The biological characteristics of these strains, including growth rate, sporulation, germination rate and timing of sporulation, were further investigated. The results showed Mf82 was the best strain having strong pathogenicity and exhibited a great potential for sustainable control of N. lugens.
  • Mycosystema. 2012, 31(3): 341-349.
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    To develop a simple and feasible way to screen high virulent fungal pathogen against insect, culture characteristics, especially the phenotypes of synnema formation, pathogenicity against Plutella xylostella larvae, and genetic variation of 5.8S-ITS rDNA sequences were evaluated for the Isaria cateniannulata isolates collected from different geographical regions. Culture characteristics of the isolates tested displayed three different types, dense synnema, loose synnema and no synnema. The type with dense synnema had high pathogenicity with over 88.9% of larva mortality rate. The highest mortality rate of the insect (98%) was caused by the isolate XS.1 in this type. The next best was the type with loose synnema, which resulted in a 68.4% of the mortality. Only 35% of larva mortality was caused by the isolates in the type without synnema suggesting a low pathogenicity. Phylogenetic analysis showed that the strains with high pathogenicity such as XS.1, XS.2 and SL.7 were closely clustered to a subclade and the strains with lower pathogenicity including 8.02 and 468.10 were clustered together. Isolates 468.10 and 8.02 without synnemata were clustered in a separated subclade. These results demonstrated a distinct intraspecific genetic variation, and a relevance of the ability to produce synnemata to the virulence against P. xylostella larvae in I. cateniannulata. Thus, the ability of synnema formation could be an important factor to screen high virulence isolate of I. cateniannulata isolates.
  • Mycosystema. 2012, 31(3): 350-358.
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    The full-length cDNA of a hydrophobin gene Nrhyd gene was cloned from the insect pathogenic fungus Nomuraea rileyi using SMART RACE RT-PCR. The expression of Nrhyd gene by N. rileyi under different culture conditions was analyzed using Realtime-PCR, and the effects of different culture conditions on expression of Nrhyd gene on mRNA level were explored. Sequence analysis shows that the cDNA contains an open reading frame of 339bp which encoding a polypeptide with 111 amino acids. The precursor protein has a molecular mass of 10.6 and a calculated pI of 6.19. The RT-qPCR results showed that the expression of Nrhyd gene was suppressed under liquid culture condition. While under solid agar culture condition, the expression level of Nrhyd gene increased along with the formation of conidia and reached the highest level at day 8 of post-inoculation, and then decreased gradually. It is inferred therefore that the Nrhyd gene might play an important role in the formation of conidia in N. rileyi. Phylogenetics analysis of Nrhyd gene with other fungal hydrophobin indicates the gene is most closely related to the homologues from Metarhizium spp.
  • Mycosystema. 2012, 31(3): 359-365.
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    By screening a cDNA library, the full length cDNA sequence and DNA sequence of a cyanide hydratase gene was isolated from Metarhizium acridum. Sequence analysis showed that the MaChy gene had no intron and the open reading frame was 1,074bp which encodes a protein with 357 amino acid residues with a calculated Mr of 39.78kDa and pI of 5.53. The characters of this putative protein were analyzed by several online bioinformatics tools, including signal peptide sequence, transmembrane segment, and secondary structure. The results indicated that this protein was neither a secreted protein nor a membrane protein. The putative amino acid sequence of MaChy shared high identities with the Chy proteins from other fungi. The highly conserved catalytic triad constituted by Glu-Lys-Cys could be found in the protein. A quantative RT-PCR analysis showed that the gene expression level was highly up-regulated at the appressorium formation stage, and it was about 2.5 times over the colonization of host hemolymph stage.
  • Mycosystema. 2012, 31(3): 366-373.
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    To monitor the effect of a Metarhizium acridum strain spread in the field, a DNA molecular marker to differentiate a released strain from other strains or native isolates was developed. Sixteen arbitrary primers to amplify the genomic DNA of 51 Metarhizium strains were used and 81 polymorphic sites were obtained. Among them, 30 polymorphic bands were obtained from the strain M189. One band ascertained to be specificity to the strain M189 was sequenced and transformed as a specific SCAR (sequence characterized amplified regions) marker for this strain. The marker sensitivity was tested and confirmed by identifying strain M189 from 51examined strains. The SCAR marker was used to distinguish 3 isolates from the soil samples collected from the field and confirmed one of them being the released strain M189.
  • Mycosystema. 2012, 31(3): 374-381.
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    The volatile components of mycelia of Paecilomyces hepiali and Paecilomyces gunnii were extracted by simultaneous distillation extraction (SDE) and analyzed by gas chromatography-mass spectrometry (GC-MS). The results indicated that there were 25 and 32 volatile components in mycelia of P. hepiali and P. gunnii, respectively. Butylated hydroxytoluene and 2H-pyrrol-2-one,1,5-dihydro-1-methyl are the two major volatile components in the mycelia of P. hepiali, accounting to 65.78% and 12.77%, respectively. Butylated hydroxytoluene and (E,E)-2,4-decadienal are the two major volatile components in the mycelia of P. gunnii, accounting to 62.11% and 12.32%, respectively. Eight components are in common in liquid submerged fermentation mycelia of the two fungi and make up 71.58% and 69.67% of the total volatile components, respectively, indicating that the major volatile components in the two experimental mycelia are similar. The analysis of volatile components provided a better understanding of the pharmaceutical activities of the mycelia of P. hepiali and P. gunnii.
  • Mycosystema. 2012, 31(3): 382-388.
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    In order to screen high-yield strain and determine the best harvest time of cordycepin, 14 Cordyceps militaris strains was tested by shaking flask fermentation. The effect of 8 different kinds of nutrient additives on the dynamics of cordycepin accumulation were compared. The results showed that C. militaris CM001 strain produced the highest amount of cordycepin. The biomass of C. militaris reached the maximum of 20.44mg/mL at the 10th day while the maximum cordycepin content reached 73.81mg/L at the 13th day. Seventy percent of cordycepin was in the fermentation broth. Additions of adenosine, adenine, alanine, glycine, and L-aspartic acid considerably increased the cordycepin production in the submerged culture, especially the addition of 1g/L adenine which increasing the cordycepin production by 7.09-fold than the control.
  • Mycosystema. 2012, 31(3): 389-397.
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    The medicinal fungus Cordyceps militaris produces many active metabolites including cordycepin (3¢-deoxyadenosine). The effects of additives and different liquid culture conditions on cordycepin production by C. militaris were investigated. The strain of C. militaris 08Y1 was used for optimizing liquid culture conditions (light and shaking, light and static, dark and shaking, dark and static), and the additives (adenine 1g/L+glycine 16g/L) were added to medium for 16 days. The maximum cordycepin production of 1,015.0mg/L was achieved by culture at dark and shaking with the supplement of additives, and the maximum utilization ratio of adenine reached 98.5%. The results showed that the condition of dark and shaking with the supplement of additives was an effective way for increasing the production of cordycepin.
  • Mycosystema. 2012, 31(3): 398-404.
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    The liquid fermentation kinetics of Metarhizium anisopliae IMI330189 was observed. Kinetic models of biomass growth and substrate consumption were established based on Sigmoid model and the parameters of the models were determined by Origin7.5 software. The results showed that the models fitted well with the liquid fermentation process of M. anisopliae IMI330189. The maximum specific growth rate was found to be 0.084h-1 at 22.8h, specific total sugar consumption rate 0.246h-1 at 9.6h and specific total nitrogen consumption rate 0.007h-1 at 10.3h. The maximum yield coefficient of biomass from total sugar was 0.861g/g at 39.8h. The model simulation matched well with the experimental observations, which made it possible to elucidate the kinetic characteristics of M. anisopliae IMI330189 during liquid fermentation. These models thus can be employed for the development and optimization of M. anisopliae IMI330189 liquid fermentation processes.
  • Mycosystema. 2012, 31(3): 405-412.
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    Spectrophotometer was used to examine different extraction method of adenosine components from anamorphic mycelia of Cordyceps species. Thin layer chromatography and high performance liquid chromatography were used to analyze qualitatively and quantitatively the adenosine components including adenine, adenosine, cordycepin and N6-(2-hydroxyethyl)-adenosine (HEA). The results showed that using distilled water ultrasonic treatment for 30min was the best extraction method. The data showed that five examined species of Cordyceps could produce adenosine and adenine. Cordyceps militaris produced cordycepin, and Cordyceps pruinosa and Cordyceps cicadae produced HEA. It was found that the incubation duration could affect the accumulation of HEA in the anamorphic mycelia.
  • Mycosystema. 2012, 31(3): 413-421.
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    Liquid culture conditions and extraction process of ergosterols from the entomopathogenic fungus Paecilomyces gunnii were optimized by using the indices of biomass and ergosterol productions. The result of orthogonal test indicated that the optimal liquid medium contained sucrose 4%, bran 2%, KH2PO4 0.025% and NaCl 0.05%. Response surface experiment demonstrated that the optimal extract solvent concentration was 80% ethanol, the ratio of solvent to raw material was 200:1 and the extract duration was 65min.
  • Mycosystema. 2012, 31(3): 422-431.
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    The factors of carriers, accelerant additives and water addition were the key factors in granule formulation processing of Metarhizium anisopliae. After study the influence of these factors on conidial viability, we found that ten examined carriers had no significantly negative effects on conidial germination. Of these, on the contrary, the calcium bentonite, kaolin, pulvistalci, floridin and light calcium carbonate could improve the shelf-life of fungal conidia. Two types of accelerant agents, i.e., trehalose and tyrosine, could significantly improve the conidia germination with varying rates at 15.12%-24.88% and 15.06%-29.67%, respectively. The effects of water addition on granulation ratio and granulation temperature were obviously correlated. By regression analyses, the suitable addition of water was optimized for reaching more than 90% granulation ratio and obtaining corresponding granulation temperature. The suitable additional quantity of water and suitable granulation temperature for calcium bentonite were 19.1%-25.0% and 50.2-54.5℃, for kaolin 35.0%-40.0% and 55.1-56.2℃, for pulvistalci 27.2%-40.0% and 50.4-51.1℃, for floridin 18.3%-25.0% and 51.3-53.6℃ and for light calcium carbonate 40.8%-45.0% and 49.7-56.1℃, respectively. The shelf-life of formulated conidia could be effectively prolonged during storage.
  • Mycosystema. 2012, 31(3): 432-442.
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    Cordyceps guangdongensis is a species found in South China not long ago. It has been successfully cultivated. Previous studies indicated that the fruiting body of the fungus has noticeable effects of anti-oxidation, anti-H9N2 virus, anti-fatigue and life-prolonging, but its effect on treatment of renal disease has not yet been reported. In this study, therapeutic effects of the fungus on chronic renal failure (CRF) rats were tested. The CRF rat model was induced by feeding adenine, and the rats were divided into 6 test groups, a blank control group without any medicine treatment, a positive control group treated with a Chinese patent medicine ‘Niaoduqing’, a normal control group of healthy rats and 3 treatment groups fed respectively with low dose, middle dose or high dose of the fruiting body of C. guangdongensis. The levels of rat blood urea nitrogen (BUN), creatinine (Cr), amount of emiction within 24h, urinary protein (UP) and pathological reaction of nephridial tissue were rountinely investigated with Creatinine (Cr) kit, blood urea nitrogen(BUN)kit, albumin (ALB) kit, total protein (TP) kit and urinary protein (UP) kit and standardized approaches. The results were analyzed through comparisons of the tested data from different groups of CRF rats as well as from the normal control group of healthy rats. The results showed that feeding C. guangdongensis fruit body could improve the renal functions and reduce the BUN and Cr of the CRF rats, promote the body to generate albumin (ALB) and total protein (TP) to make up the proteins that were partially lost owing to renal damage, and alleviate the renal pathological changes of CRF rats. The conclusion can be drawn that the fruiting body of C. guangdongensis has prominent therapeutic effects on CRF rats.
  • Mycosystema. 2012, 31(3): 443-449.
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    There are significant differences of the polysaccharide content from Cordyceps militaris assayed with different methods in previous literatures, which could be mainly caused by removing the interfering substances or not. After monitoring the interfering substances by high performance anion exchange chromatography, two common analysis methods for determination of the polysaccharides of C. militaris were compared. We found that the extraction with 80% ethanol in twice could effectively remove the interfering substance. Our study showed that it was a sensitive, accurate and reproducible method and could be successfully applied to analyze the water soluble polysaccharides produced by C. militaris.
  • short communications
  • Mycosystema. 2012, 31(3): 450-457.
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    Beauveria bassiana strain FDB01 was isolated from naturally diseased pine sawfly, Acantholyda parki, collected from Yitong of Jilin Province in the northeast of China. Its pathogenicity was tested with conidial suspension (2×108conidia/mL) on the two species of pine sawfly, Acantholyda posticalis and Pristiphora erichsonii. The results showed that the strain FDB01 was pathogenic fungus and caused the death of A. parki. Treatment with conidial suspension the mortalities of the larvae of A. posticalis and P. erichsonii reached 94.4% and 100%, respetively in 16d. It indicated that FDB01 is a high virulent strain to pine sawflies.