One new species, Pseudocercospora hunanensis on Diospyros kaki, and 2 new records for China, Cercospora pseudokalanchoës on Sedum spectabile, and Pseudocercospora piperina on Piper sarmentosum are reported. Descriptions, illustrations, discussions are provided. Examined specimens are deposited in HMAS.
Two Aspergillus strains (BC-D1 and BC-212) were isolated from dead and mycosed adults of the melon fly Bactrocera cucurbitae in Hainan. The strain BC-D1 was identified as Aspergillus flavus and the strain BC-212 was Aspergillus tamarii based on their morphological characters and the rDNA-ITS fragment sequences. The virulence of BC-D1 and BC-212 against B. cucurbitae was tested in laboratory. The results indicated that two Aspergillus species had higher virulence against the adults of B. cucurbitae than against insect larvae, pupae and egg. The mortalities of adults in 8d after inoculation with BC-D1 and BC-212 were 73.5% and 85.1%, respectively. However, mortalities of the larvae, pupae and egg were less than 50%. Both species caused very low mortalities against the larvae of Tenebrio molitor, Spodoptera litura, and Lipaphis erysimi. They were characteristed by rapid growth and high sporulation. The optimum growth temperature were 30-35°C. With the difference of nutritional composition in medium, the growth rate, sporulation and morphology features were comparatively different from each other.
A habitat survey of autumn-occurred morels was conducted in Yanyuan, Huidong and Mianning counties in Liangshan Yi Autonomous Prefecture, Sichuan, China and 11 specimens were collected. By morphological characteristics and DNA sequences of ITS, LSU, ef1-α, rpb1 and rpb2, the samples were identified as Morchella septimelata (Mel-7) and Morchella sp. (Mes-16). Morchella septimelata was first reported in Sichuan Province. The suitable environment for autumn-occurred morels is forest soils on steep slopes in mountainous region.
Arbuscular mycorrhizal fungi (AMF) can effectively improve plant fitness and soil quality, thus they play an essential role in soil remediation and revegetation in these degraded ecosystems. Using cloning and sequencing methods, we examined the AM fungal assemblages in the rhizosphere of Bauhinia faberi var. microphylla which is a dominant shrub species in the dry valley of Minjiang River of Sichuan Province for facilitating theoretical studies on preservation and recovery of native vegetation. We detected 53 AMF operational taxonomic units (OTUs) which were assigned to 7 families and 10 genera of Glomeromycota based on phylogenetic analysis of sequences. Glomus, Diversispora and Funneliformis were dominant genera in the rhizosphere of the plant. Glomus taxa were most abundant, accounting for more than 50% of the total OTUs and sequences, respectively. Rhizophagus and Archaeospora were relatively rare, and the rest were common genera. Our results reveal that there is a high level of AMF diversity in the rhizosphere of the target plant species, and these abundant AMF taxa may play an important ecological role in this arid region.
The population diversity of Phytophthora infestans, the most destructive pathogen causing potato late blight, in Sichuan Province were identified by phenotypic and genotypic analysis. The mating type, metalaxyl sensitivity and virulence test were included in phenotypic analysis. Amongst 43 isolates tested, two were A1 mating type, 29 were A2 mating type and 12 were self-fertile (SF). The results of metalaxyl resistivity test indicated that three isolates were resistant, 22 were moderately resistant and eight were sensitive. Eleven virulence types were identified, among which 25.58% of the isolates showed full virulence with the highest frequency. Based on genotypic analysis, ten SSR genotypes were found and 16 alleles were yielded in 43 isolates with an average of 3.2 alleles per locus. The mean PIC value was 0.46. SSR4 was the most informative SSR, with the highest PIC value (0.76). A2 and SF isolates were grouped to the same cluster. Full virulence races were dominant in local potato fields. These results provide a scientific basis for controlling potato late blight in Sichuan Province.
Metarhizium robertsii, a filamentous ascomycete fungus, is well known as the causative agent of insect green muscardine disease. By homologous genes blast, a single copy of pyrimidine precursor biosynthesis enzyme gene MAA_02402, named MrThi12, was found in the genome of M. robertsii. The MrThi12 cDNA has a full length of 1 029bp, which encodes a protein of 342 amino acid residues. This study aimed at characterizing the function of MrThi12 gene by knocking it out using the method of target gene replacement. The ΔMrthi12 mutant strain grew very slowly, and could not produce aerial hyphae and conidia in the vitamin B1-lacking media. However, when vitamin B1 was added in the medium, the phenotype of the ΔMrthi12 mutant strain could be fully recovered, without significant difference with the wild type strain.
Fruiting bodies of Hericium erinaceus harvested at seven different growth stages were extracted by hot water and graded ethanol precipitation to final concentration of 20%, 50% and 70%, and 21 crude polysaccharide fractions were obtained. Then structural properties and immunity activities in vitro of polysaccharides were studied. Results showed that in the whole developmental stages of H. erinaceus fruiting bodies, the overall yield of polysaccharides appeared an increasing trend at first then decreasing after the fifth stage. At fifth stage the maximum yield was 0.92%. Polysaccharide content of 20% ethanol precipitation also showed an increasing trend at first then decreasing, and the maximum polysaccharide content was 42.87% at the fifth phase, in which high molecular weight polysaccharides (1 000-5 000kDa) had the highest proportion. Polysaccharide molecular weights of 50% and 70% alcohol precipitation were all small, ranging from 10 to 40kDa. Most of the H. erinaceus polysaccharides obtained consisted of fucose, galactose, glucose and mannose, while some differences in the relative proportions existed. The polysaccharides of 20%, 70% ethanol precipitations contained a small amount of ribose at the first stage and 20% ethanol precipitation contained some rhamnose at the seventh stage. All stages of 50% alcohol precipitation polysaccharides contained a certain amount of glucuronic acid. All the 21 fractions of polysaccharide could significantly stimulate macrophages to release NO in vitro, and activity of polysaccharides of 20% ethanol precipitation was better than that of 50% and 70% ethanol precipitation, which showed good immune activity in vitro at 50μg/mL. Results showed that the activities of polysaccharides at the fifth stage were best, indicating best medicinal and health protection effects, and the polysaccharides at this stage could be regarded as the best materials for production of health products. Our findings provided a theoretical basis for the study of polysaccharide dynamic formation during the development of H. erinaceus fruiting bodies.
A method of high performance liquid chromatography (HPLC) was established to carry out qualitative and quantitative analysis of fifteen nucleosides in Ganoderma lingzhi spore powder. HPLC was performed on Ultimate AQ-C18 (4.6mm×250mm, 5μm). The mobile phase consisted of methanol and water with gradient at a flow rate of 1.0mL/min. The UV detection wavelength was set at 259nm, the column temperature was 30˚C, and the sample volume was 10µL. The methodological study showed that the method was sensitive, accurate, reproducible and reliable, thus it was available to analyze the content of the nucleosides in the G. lingzhi spore powder. Composition and content of 15 nucleosides of wall-unbroken spore powder harvested in different harvest period from four producing areas, Longquan, Fenghua, Dabieshan, and Huangshan, were determined with designed method above-mentioned. The results indicated that the wall-breaking treatment duration had a mild influence on the extraction yield of nucleosides. The nucleosides in the spore powder from different areas were significantly varied in their composition and content. The content of the nucleosides increased with the extension of duration of harvested period. Cytosine, uridine, adenine, guanosine and adenosine were contained in all the samples. The content of uridine, guanosine and adenosine accounted for over 70% of the total nucleoside content in all the samples, indicating that they were the main nucleosides in the G. lingzhi spore powder.
One-factors-at-a-time experiments were carried out to investigate the effects of sterilizing condition, incubation duration, selenium addition amount and optimal replenishing time on the yield of crude intracellular polysaccharide of selenium-enrichded Lyophyllum decastes mycelia. The process parameters were optimized using a Box-Behnken experimental design coupled with response surface methodology. Culture medium and sodium arsenate solution were mixed after respective sterilization for 20 minutes under 121˚C. It was showed that addition of 4µg/mL Na2SeO3 in 129h of incubation and continued extension of incubation duration resulted in high yield of crude intracellular polysaccharide in Lyophyllum decastes mycelia, reaching 349.8mg/100mL in 244h of fermentation.
A kinetic model of batch fermentation for penicillin G acylase (PGA) enzyme production by recombinant Pichia pastoris was established. The influences of glycerol consumption, methanol concentration, Pichia pastoris concentration, dissolved oxygen and feeding time on the PGA activity were investigated in batch fermentation process. Using matlab software, the kinetics model for cell growth with multisubstrates, PGA production and substrate consumption was established through optimal parameter estimation and nonlinear fitting. The model calculation fitted well with the experimental observations, and showed that the constructed kinetic models could well reflect the batch fermentation for PGA production by recombinant P. pastoris.
β-glucans from Ganoderma lingzhi fruiting bodies were extracted using ethanol-enzyme pretreatment combined with the typical water extraction method. The results showed that the key parameters of ethanol-enzyme pretreatment system were 60% of ethanol (V/V), 1.5% of enzyme (M/V, g/mL), 45˚C of enzymolysis temperature and 8.0 of enzymolysis pH. Based on the ethanol-enzyme pretreatment method, the hot water extraction technology of β-glucans was further optimized using single factor test and Box-Behnken test design. The results revealed that the key factors, extraction temperature, extraction time, ratio of water to material, and the interation effect between extraction temperature and ratio of water to material had significantly affected on the extraction of β-glucan from G. lingzhi. The optimal levels of the three factors were temperature 80˚C, extraction duration 2.5h and ratio of water to material 35:1. Under these conditions, the yield of β-glucan from G. lingzhi was up to 0.412mg/g being 1.1 times higher than that by the traditional ethanol pretreatment combined with typical water extraction method. This study provides an useful technology for β-glucan extraction from G. lingzhi fruiting bodies and lays the technical basis for β-glucan extraction in a large scale in future.
A new leaf spot disease was observed on strawberry Fragaria ananassa in Fangshan District, Beijing in 2013. Isolation of the pathogen was made from symptomatic tissues and isolate CMF4 was obtained. The pathogenicity and host range was examined by inoculation in laboratory. The generated strain CMF4 was able to infect the damaged leaves and fruits of strawberry. Its host range test showed that it could infect the damaged leaves of peony Paeonia suffruticosa, Chinese flowering crabapple Malus spectabilis, Chinese herbaceous peony P. lactiflora, apricot Armeniaca vulgaris, cherry Cerasus pseudocerasus, peach Amygdalus persica and Chinese rose Rosa chinensis, but failed to infect their non-damaged leaves, and the similar fungus could be isolated from these diseased plants. Based on rDNA-internal transcribed spacer (ITS) analysis and morphological characters, the pathogen was identified as Pestalotiopsis clavispora. The pathogen was firstly reported in China responsible for Pestalotiopsis leaf spot of strawberry.
