Review on Research Progress and Prospects of Cicada Flower, Isaria cicadae (Ascomycetes).

Cicada flower, Isaria cicadae Miq., has been a traditional Chinese medicine for approximately 1600 years. Many works on its identification, bioactivities, and clinical use against some disorders have been published, but some inaccuracies and inconsistencies need to be further clarified. In combination with our > 20 years of research and application of cicada flower and examination of the literature and patents published in recent years, this article summarizes and reviews the life cycle and taxonomy, genome size and mating type, molecular systematic classification and cultivation, active ingredients, and pharmacological functions of I. cicadae.

[Population genetic structure of Isaria cicadae causing enzootic of cicadas nymphs]. / å¼•è‡´è‰è‹¥è™«åœ°æ–¹ç— çš„è‰æ£’æŸå­¢ç§ç¾¤é—ä¼ ç»“æž„.

The population genetic structure of Isaria cicadae, which caused enzootic of cicadas nymphs in three regions, was analyzed by ISSR marker. The results showed that all three enzootic populations showed high genetic diversity with the highest in the Jingtingshan population and the lowest in the Shitai population. The UPGMA clustering analysis revealed that different enzootic populations did not have a predominant lineage but were polyphyletic and heterogeneous. Genetic lineages had nothing to do with geographical origin. However, two subpopulations of Jingtingshan from different sampling periods were gathered into different clades, which exhibited remarkable temporal heterogeneity. The genetic differentiation (Gst) among populations (subpopulations) was 0.2153 and the gene flow was low at 0.9110 (Nm＜1), which indicated the low gene flow was one of the main reasons for the genetic variation in the population. Therefore, high heterogeneity and low dominance might be genetic structure characteristics of I. cicadae population causing enzootic of cicada nymphs.

Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD50) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 µg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time.

[Genetic diversity and population genetic structure of Beauveria bassiana in northern China].

A total of 622 isolates of Beauveria bassiana collected from 13 provinces in northern China were divided into 13 provincial subpopulations, and 568 of the 622 isolates belonging to 8 insect orders and Araneida, whose hosts could be indentified to order level, were divided into 9 host order subpopulations, with the genetic diversity and population genetic structure of the B. bassiana in northern China analyzed by using ISSR (inter-simple sequence repeat) technique. All the diversity indices showed that the B. bassiana in northern China had higher genetic diversity and population heterogeneity. Among the B. bassiana subpopulations, Inner Mongolia subpopulation and Lepidoptera subpopulation showed the highest genetic diversity and population heterogeneity, while Henan subpopulation and Araneida subpopulation showed the lowest ones. The genetic differentiation index and genetic distance between Henan and Liaoning subpopulations and between Araneida and Mantodea subpopulations were the highest, while those between Ningxia and Shaanxi subpopulations and between Coleoptera and Hymenoptera subpopulations were the lowest. The mean genetic differentiation index and mean genetic distance between the host order subpopulations were lower than those between the provincial subpopulations. These results and the subpopulation clustering analysis based on the genetic distance of subpopulations all demonstrated that the genetic lineage of B. bassiana isolates in northern China was associated with neither their geographic origin nor their host origin. The variation of B. bassiana in northern China was mainly caused by the variation between families and between genera of different host orders, and also, caused by the diversity between different collection sites and between different microhabitats within the collection sites.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

[Effects of Beauveria bassiana on Myzus persicae and its two predaceous natural enemies].

A Beauveria bassiana strain Bb21 was isolated from naturally infected green peach aphid Myzus persicae (Hemiptera: Aphididae). The effects of the strain on M. persicae and its two predaceous natural enemies Chrysoperla carnea (Neuroptera: Chrysopidae) and Harmonia axyridis (Coleoptera: Coccinellidae) were investigated under laboratory conditions. Bb21 had strong pathogenicity to M. persicae, with the LD50 of 97 conidia x mm(-2) (45-191, 95% confidence interval), but was less pathogenic to the second instar nymph of C. carnea, with the LD50 of 1089 conidia x mm(-2). The LD50 for C. carnea was 10.2 times higher than that for M. persicae. The pathogenicity of Bb21 to H. axyridis was very weak, with a low infection rate of 13% even at a high concentration 5 x 10(8) conidia x mL(-1). The Bb21 at low conidia concentration had less effect on the developmental period and fecundity of the two predaceous natural enemies. However, when applied at the high concentration 5 x 10(8) spores x mL(-1), Bb21 shortened the larval stage of H. axyridis averagely by 1.4 d and decreased the adult emergence rate and fecundity by 33% and 14%, respectively, and shortened the larval stage of C. carnea averagely by 0.7 d and decreased the adult emergence rate and fecundity by 24% and 11%, respectively. Since the LD50 for green peach aphid was much lower than that for the two predaceous natural enemies, and had very low effect on the adult emergence rate and fecundity of the two predators at the concentration recommended for field spray, Bb21 could be applied as a biocontrol agent of M. persicae in the integrated management of pernicious organisms.

[Niche comparison of dominant entomopathogenic fungi in three forest ecosystems].

An investigation was made on the quantitative composition, niche width, and niche overlap of dominant entomopathogenic fungi in three different forest ecosystems, i.e., natural broad-leaved forest, natural secondary broad-leaved forest, and pure Masson' s pine plantation. In the three forest ecosystems, Beauveria bassiana was the first dominant species in natural secondary broad-leaved forest, the second in pure Masson's pine plantation, and the third in natural broad-leaved forest. B. bassiana had the broadest temporal niche width and nutritional niche width, whereas the dominant species Isaria cateinannulata, L. farinose, and I. tenuipes had much smaller niche widths. Meanwhile, B. bassiana had larger temporal niche overlaps but smaller nutritional niche overlaps with other dominant entomopathogenic fungi. It was suggested that in the three forest ecosystems, B. bassiana had the longest occurrence duration, widest host range, and strongest environmental adaptability.

[Genetic differentiation of Isaria farinosa populations in Anhui Province of East China].

Isaria farinosa is an important entomopathogenic fungus. By using ISSR, this paper studied the genetic heterogeneity of six I. farinosa populations at different localities of Anhui Province, East China. A total of 98.5% polymorphic loci were amplified with ten polymorphic primers, but the polymorphism at population level varied greatly, within the range of 59.6%-93.2%. The genetic differentiation index (G(st)) between the populations based on Nei's genetic heterogenesis analysis was 0.3365, and the gene flow (N(m)) was 0.4931. The genetic differentiation between the populations was lower than that within the populations, suggesting that the genetic variation of I. farinosa mainly come from the interior of the populations. The UPGMA clustering based on the genetic similarities between the isolates revealed that the Xishan population was monophylectic, while the other five populations were polyphylectic, with the Yaoluoping population being the most heterogenic and the Langyashan population being the least heterogenic. No correlations were observed between the geographic distance and the genetic distance of the populations. According to the UPGMA clustering based on the genetic distance between the populations, the six populations were classified into three groups, and this classification was accorded with the clustering based on geographic environment, suggesting the effects of environmental heterogeneity on the population heterogeneity.

[Population genetic structure of Beauveria bassiana from south and southwest Anhui sericultural regions: ISSR analysis].

White muscardine caused by Beauveria bassiana is an important factor affecting sericulture. In the present study, a total of 124 B. bassiana isolates were obtained from the silkworm cadavers, rearing rooms and appliances, surrounding mulberry orchards, pine plantations, and croplands in Jingxian County of south Anhui and Qishan County of southwest Anhui. Together with the mass production strains, the isolates were analyzed for population genetic structure by ISSR markers to trace the origin and the spreading track of the muscardine. The results showed that the two B. bassiana populations in Jingxian County and Qishan County were heterogenic, being monophyletic in Jingxian and polyphyletic in Qishan. Both the Jingxian population and the Qianshan predominant subpopulation were characterized typically by enzootic nature, i.e., low incidence and frequent occurrence, but the Qianshan non-predominant subpopulation could spread among some alternate hosts outside the rearing rooms. The groups prevailing in pine caterpillar populations in the pine plantations of Qianshan and Jingxian, the production strains, and the epizootic strain prevailing in surrounding mantids were all not associated to the silkworm muscardine, displaying safety to sericulture.

[Genetic heterogeneity of Beauveria bassiana population in pure Masson's pine plantation].

By the method of ISSR, the genetic heterogeneity of 111 Beauveria bassiana isolates in a pure Masson' s pine plantation in Magu Mountains of Anhui Province was analyzed. A total of 58 loci were obtained with 7 ISSR primers, 54 of which (93.10%) were polymorphic. For the isolates sampled at different time, their Nei's gene diversity (He) was 0.2552, Shannon's diversity index (I) was 0.3825, coefficient of gene differentiation (Gst) was 0.2269, and gene flow Nm was 0.8518. The genetic distance between the isolates sampled in May and July was the shortest (0.0408). For the isolates grouped based on host orders, their He was 0.2623, I was 0.3884, Gst was 0.1964, and Nm was 1.0223. The genetic distance between the isolates from Coleopteran and Hymenoteran was the shortest (0.0163). All the results suggested that the genetic heterogeneity of B. bassiana population in the test plantation was rich, and the genetic variation between B. bassiana sub-populations was relatively low but that within the sub-populations was high, and none of the isolates were identical or highly similar with each other.

[Species diversity and seasonal change of entomogenous fungi in Langya Mountains Nature Reserve].

672 specimens of entomogenous fungi belonging to 20 species, 8 genera and 4 families were collected from 20 sampling quadrates in the Langya Mountains Nature Reserve of East Anhui. The dominant species were Beauveria bassiana, Paecilomyces farinosus, P. cateinannulatus, B. brongniartii and P. fumosoroseus, and B. bassiana was most dominant, with the relative abundance of 73.8%. The entomogenous fungi in the Reserve were high in amount but low in diversity. The amount of isolates and the abundance of each species were the highest in summer and decreased with decreasing air temperature and rainfall, while the Pielou evenness changed from relatively low to relatively high. Paecilomyces spp. had a less seasonal fluctuation of quantity than Beauveria spp.

[Molecular evaluation on the efficacy of sustainable control of Monochamus alternatus by non-woven fabric fungal bands impregnated with Beauveria bassiana].

By using molecular marker of group I intron, a field evaluation was made on the efficacy of sustainable control of Monochamus alternatus by non-woven fabric bands impregnated with Beauveria bassiana. The results showed that the control efficacy in two treated plots was 19.5% and 10.8%, and 14.2% and 11% when evaluated with conventional and molecular evaluation methods, respectively, suggesting that the conventional method which evaluated the control efficacy in terms of microbes mortality or infection rate couldn't distinguish the efficacies caused by released biocontrol agents and indigenous pathogens, while molecular method could avoid the interference of indigenous pathogens and give an objective and reasonable evaluation on the efficacy of microbial control.

[Genetic diversity of Beauveria bassiana (Bals.) Vuill. in forest ecosystem assessed by inter-simple sequence repeat (ISSR) markers].

In the present paper, the genetic diversity of 48 Beauveria bassiana strains from different altitudes and at different seasons in Dabie Mountains of western Anhui was estimated using inter-simple sequence repeat (ISSR) markers. Twelve among 33 ISSR primers were chosen for their reproducibility and high polymorphism. Seven (2 - 11) markers per primer were scored, and a total of 84 fragments were amplified, in which 73 (81%) were polymorphic. Genetic diversity analysis revealed a relatively high level of intraspecific genetic diversity of B. bassiana in Dabie Mountains of western Anhui: the percentage of polymorphic loci (PPL) was 81%, Nei's genetic diversity (He) was 0.3187 and Shannon's genetic diversity index (I) was 0.4782. The genetic differentiation, Gst was 0.1028, indicating that a low degree of genetic differentiation occurred in the B. Bassiana among populations.