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DISCUSSION

Contact angle is a commonly used indicator to measure the degree of surface hydropho­bicity, surface energy, heterogeneity, and roughness (31). Chau et al. (32) evaluated that the water contact angle of fungi was greater than 90°, and the fungi showed hydropho­bicity; and the larger the contact angle, the stronger the hydrophobicity. The results of this study showed that the contact angle of C. chanhua under aseptic culture and soil-covered culture was greater than 90°, indicating the hydrophobicity of C. chanhua membrane, and it is the key to its survival and adaptation to the environment. The surface hydrophobicity of fungi enables them to better adsorb organic substances (33), which can be used as one of the surface characteristics affecting the adhesion and transportation of fungi in heterogeneous media (34). Fungi reproduce by producing hyphal networks and secrete enzymes to degrade complex nutrients into monosacchar­ides to maintain cell growth (35). The secretion of hydrophobin can significantly reduce the surface tension, enabling the mycelium to escape from the liquid and grow into the air (36). Fungal surface hydrophobic proteins are involved in various functions of fungal growth and development and help conidia adhere to insect epidermis (37), which has been reported in Metarhizium anisopliae and Beauveria bassiana (38, 39).

Through scanning electron microscope observation, it can be seen that the sterile-cultivated C. chanhua mycoderm is closely arranged and the soil-covered C. chanhua mycoderm is relatively sparse and disordered. This is because the soil-covered C. chanhua has certain resistance to growth in the soil, while the sterile-cultivated C. chanhua can grow in the air. Previous studies have observed that there were H-type hyphal fusions among the mycoderm hyphae of C. sinensis (40). Interestingly, we also observed that a large number of H-type hyphal fusions were also observed in the mycelia of C. chanhua. Therefore, there are a large number of H-type hyphal fusions in the mycelia of entomo­pathogenic fungi, and H-type hyphal fusions are often closely related to parasexual reproduction (41). Parasexual reproduction is widespread, which is a special reproduc­tive mode occurring in fungi and an important source of fungal genetic variation (41, 42). Through parasexual reproduction, the genetic material between individuals can communicate with each other, which is more convenient for the exchange of genetic material between individuals, which is easier for fungi to obtain and establish population advantages than sexual reproduction (43). In a complex environment, the formation of fungal mycoderm is affected by many factors, such as the roughness of the attachment surface, environmental factors, hydrophobicity, and other factors (44). In this study, scanning electron microscopy was used to observe the arrangement of the hyphae of C. chanhua and calculate their hyphal density. Our research results showed that the bacteria-membrane mycelia of aseptically cultivated C. chanhua were closely arranged, and the growth density of mycelia was larger than that of soil-covered C. chanhua. While the mycelia of soil-covered C. chanhua were relatively sparse and disorderly, the density was smaller, which was speculated to be related to the growth environment. There is a certain resistance to the growth of soil-covered C. chanhua in the soil, but the sterile-cultivated cicadas can grow smoothly in the air, so the mycelia are closely arranged. We can continue to explore the influence of different environmental factors on the formation of C. chanhua membrane in the future, which will help us understand the adaptation mechanism of C. chanhua to the environment and promote the ecological simulation of C. chanhua.

This study found that different cultivation environments had a greater impact on the composition and diversity of the bacterial community of C. chanhua; the  finding indicated that external microbial factors had a greater impact on the bacterial commun­ity composition of entomopathogenic fungi. The common genera of all C. chanhua samples were analyzed, and the common bacteria included 22 genera, such as Achromo­bacter, Serratia, Pseudomonas, Silesia, Enterococcus, and Bacillus, indicating that these bacterial groups played an important role in the growth and development of C. chanhua and these bacterial groups were also found in wild C. chanhua samples (29). Previous studies have reported that the competition between microorganisms can promote the production of new active substances or increase the content of existing metabolites (45–47). For example, the production of HEA in C. chanhua was significantly increased after co-culturing bacteria of the genera Cedecea, Serratia, and Enterococcus with C. chanhua (48). In addition, studies have shown that Serratia not only has antibacterial activity but also has pathogenicity to some insect leaves (49, 50), so it is speculated that this kind of bacteria may participate in the process of C. chanhua infecting the host.

In this study, we found that with the growth of C. chanhua, more and more OTUs were found in the membrane of C. chanhua. It is reported that bacteria can move with the help of fungal hyphae (51, 52), so it is inferred that some bacteria in the mycoderm may come from the inner sclerotia. As the interface between C. chanhua and the environment, the mycoderm is a direct contact (29). We found that when the C. chanhua matured, the bacterial community on the mycelium was higher than the inner sclerotium. Therefore, it is speculated that the mycoderm may act as a barrier to prevent foreign bacteria from entering the inner sclerotia, thus playing a role in protecting C. chanhua. Based on it, we used C. chanhua mycoderm as a filter medium to filter soil bacteria and verified its biological filtering effect on bacteria. According to our results, only a small amount of bacteria grew in the soil suspension filtered by the mycoderm after the culture dish was coated, indicating that the mycoderm could filter most of the bacteria in the soil. A large number of environmental bacteria have been reported to colonize the mycoderm surface of C. chanhua (3). Although C. chanhua mycoderms grow in a complex soil environment, they can protect themselves from external pathogens, which is related to the characteristics of bacterial mycoderm (24). This mycorrhizal sheath structure with similar characteristics can enhance the disease resistance and stress resistance of host plants by absorbing and storing nutrients and effectively isolating pathogens (53). We found that C. chanhua mycoderm has a biological filtering effect on soil bacteria for the first time, which is helpful in understanding the relationship between microorganisms in the internal and external environment of C. chanhua and is of great significance for C. chanhua cultivation.

It is widely known that plants can obtain nitrogen in phytophagous insects through entomopathogenic fungi (54), and Behie et al. (55) found that M. anisopliae, B. bassiana, and Lecanicillium lecanii all showed the ability to transfer nitrogen to plants. In addition, Metarhizium robertsii has been reported to transfer nitrogen from the larvae of the wax borer to the plants of switchgrass for nitrogen fixation after infecting the larvae of the wax borer (56). It is interesting that the hyphae can transfer nitrogen from the insect body to the external environment and also can transfer nitrogen from the soil to the insect body. It has been reported that the entomopathogenic fungus C. chanhua is closely related to mycorrhizal fungi, which can transport nitrogen (28, 56). Previous studies have found that the mycoderm hyphae of entomopathogenic fungi can transfer nutrients from insects (57), and entomopathogenic fungi can also transfer nitrogen from infected insects to plants (58). Some entomopathogenic fungi, such as M. anisopliae, B. bassiana, and Verticillium cerasus, have the ability to transfer nitrogen (59). It has been reported that plants can obtain insect nitrogen sources by combining them with an endophytic entomopathogenic fungus (59). Behie and Bidochka (60) showed that all M. anisopliae and B. bassiana strains can transfer a large amount of insect-derived nitrogen to plants. C. chanhua is rich in nitrogen and during its growth and development, it will remove excess nitrogen from its body to ensure normal growth (61, 62).

In summary, this study is the first to verify that the membrane of C. chanhua has a filtration effect on bacteria and a transport function for nitrogen. In future studies, we will further explore which microorganisms C. chanhua can filter out and the  specific forms that can transport nitrogen. This will help to understand the element network distribution mechanism of C. chanhua and provide a theoretical basis for the ecological cultivation technology of C. chanhua.

Conclusion

Our study demonstrated for the first time to our knowledge that the C. chanhua mycoderm of soil-covered cultivation and sterile glass bottle cultivation was hydropho­bic. In addition, C. chanhua mycoderm not only has the biological filtration function to soil bacteria, but also has the transport function to nitrogen element in the insect body and soil.

MATERIALS AND METHODS

Materials

Strain

C. chanhua strain (strain number.: GZUIFR_DJS1) is provided by the Institute of Fungal Resources, College of Life Sciences, Guizhou University.

Culture medium

Potato dextrose agar (PDA) medium is as follows: 200 g potato, 20 g glucose, 18 g agar powder, dilute to 1,000 mL with deionized water.

LB culture medium is as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 18 g agar powder, dilute to 1,000 mL with deionized water.

The above culture media were sterilized at 0.1 MPa for 30 min.

Main reagent

The E.Z.N.A.SoiL DNA Kit was provided by American OMEGA Company. The required L-Glutamine-15N was provided by Shanghai yuanye Bio-Technology Co., Ltd; the required anhydrous ethanol and isoamyl acetate was provided by Sinopharm Chemical Reagent Co., Ltd. The 15N stable isotope standard UREA4 (15N Atom% theoretical value: 0.366) was supplied by Elemental Trace Limited in the UK. Electron microscope fixativ e and PBS buffer were also used in this experiment and were provided by Xavier Biological 

Company. Osmic acid was supplied by Ted Pella Inc. Ltd., and, 15N stable isotope standard UREA4 (theoretical value of 15N atom%: 0.366), was provided by Elemental Microanalysis Ltd., UK.

Methods

C. chanhua cultivation

C. chanhua strain was inoculated on PDA medium and activated in a constant tem­perature incubator at 25°C, and 0.05% Tween 80 solution was used to prepare the target concentration of 5 × 107 conidia/mL. Pupae of Antheraea pernyi Guérin-Méneville, sourced from the Dandong City Artificial Sericulture Base (Liaoning, China), were selected as host insects (cultivation medium) for C. chanhua, referring to Zeng et al. (3) method of surface disinfection of A. pernyi: wash the surface of A. pernyi with tap water, and dry it naturally; soak in 75% alcohol in the sterile super clean table, and scrub the insect surface with cotton for about 10 s; then rinse it with sterile water, clip it into a white porcelain plate covered with sterile paper towels, and dry the surface water; and suck 0.02 mL of prepared conidia suspension, and inject it into the third ring just behind the wing of the tussah pupa (63). The inoculated silkworm pupa is clamped into a sterile glass bottle and cultured in a dark place at 25°C. The bottle is moisturized with sterile wet cotton, and the cultivation process of C. chanhua is shown in Fig. S6.

Microbial diversity of C. chanhua

A. pernyi pupa is infected by C. chanhua fungus after being cultivated in a glass bottle for 1 week to form a stiff worm (stiff worm stage). Take out the worm body, break  off the inner sclerotium sample 5 g, and put it in a sterile centrifuge tube, with the label marked Si_1. Five silkworm pupae are mixed into one sample for three replications. After being ground evenly with liquid nitrogen, it is stored at −20°C. Some of the zombies will continue to be cultivated in sterile glass bottles, and the other part will be covered with soil under the masson pine forest of Guizhou University, with a thickness of 1–2 cm. After about 20 days of continuous cultivation in glass bottles, a layer of mycoderm formed by white mycelium (mycoderm formation stage) on the surface of C. chanhua was formed. The mycoderm was gently peeled off on the ultraclean workbench with sterile tweezers and put into a 10-mL centrifuge tube, which was recorded as Ci_2. Break off the insect body, and take the internal sclerotium sample in the centrifuge tube, which is recorded as Si_2. After 45 days of continuous cultivation in glass bottles, when the fruiting bodies of C. chanhua are almost mature (C. chanhua mature), cut off the fruiting bodies, take the samples of mycoderm (Ci_3) and inner sclerotia (Si_3) as before, and store them in centrifuge tubes at low temperature. Similarly, the samples of bacterial mycoderm (Cs_2) and inner sclerotia (Ss_2) of C. chanhua cultivated with soil mulching were taken at the same time as those of C. chanhua in aseptic glass bottles during the formation of bacterial mycoderm, and the samples of bacterial mycoderm (Cs_3) and inner sclerotia (Ss_3) were collected at the mature stage for cryopreservation. All samples were collected and ground evenly with liquid nitrogen and then stored at −20°C until DNA was extracted.

The morphology of the mycelium of C. chanhua in the soil and atmospheric environment

After C. chanhua is mature, collect the fresh C. chanhua cultivated in soil covering and sterile glass bottle respectively, and take it back to the laboratory. After washing the surface soil, use sterile paper to absorb the surface water, cut off the fruiting body, and gently peel off the bacterial mycoderm with tweezers. First, mix every five C. chanhua mycoderms into a group, repeat three times, weigh with a precision balance, record the fresh weight, and then weigh and record the dry weight after drying at 60°C. Then, observe the thickness of C. chanhua mycoderm under stereomicroscope, and use a scanning electron microscope to observe the structure, density, and arrangement of C. chanhua mycoderm under soil and air environment according to the method of Jackowiak, and see Table S3 for specific steps (64). Finally, the contact angle measur­ing device was used to determine the hydrophilicity/hydrophobicity of the C. chanhua mycoderm by using the method of Huhtamäki et al. (65).

Filtration effect of C. chanhua mycoderm on bacteria

Soil was randomly dug from the ground of masson pine forests in Guizhou University (Guiyang, Guizhou) and taken back to the laboratory; 10 g of fresh soil was weighed and added into 90 mL of sterile water. It was incubated at 25°C in a shake flask for 2–3 h. It was filtered into a new sterile triangular flask with sterile gauze. The soil suspension was diluted to 10−7 in turn for standby.

Three groups of different treatments are set up in this experiment.

1. Group A: 600 mesh sterile nylon mesh cut into 7 × 7 cm2 is folded after sterilization and put into a sterile funnel. One milliliter of soil suspension is sucked each time for filtration. The filtrate is collected with a sterile centrifuge tube until 5 mL of filtrate is collected.

2. Group B: spread the C. chanhua mycoderm peeled with sterile tweezers to about 3 × 3 cm, put it into the sterile funnel, so that the mycoderm forms a concave surface at the funnel tube, then use the pipette gun to suck the soil filtrate, and slowly drop it into the concave surface of the mycoderm (the soil suspension does not exceed the concave surface), so that it can be filtered slowly. Collect about 5 mL of filtrate with a sterile centrifuge tube.

3. Control group (CK group): in the same way as Group B, sterile water was filtered by mycoderm, and 5 mL of filtrate was collected by centrifuge tube for standby.

Soil suspension stock (S0) with 10-7 concentration, nylon mesh filtrate (S1), mem­brane filtrate (S2) and membrane filtered sterile water (Sck) were absorbed and coated on the medium with a 200 μL pipette, and the bacteria were cultured at 25℃, with 3 repeats for each treatment. After 2 days of incubation, the single bacterial colony in each culture dish was counted. The experimental process of bacterial filtration by C. chanhua mycoderm is shown in Fig. S7.

15N flows from the soil to the insect body through the C. chanhua fungus mycoderm (recorded as Experiment D)

Add 10 kg fresh soil dug from the masson pine forest to each cultivation basin, balancing the humidity for 3 days, so that its physical and chemical properties are consistent. Use sterile water to prepare 100 mg/L L-glutamine-15N solution. After it is completely dissolved, shake it up. Add 500 mL of the prepared L-glutamine-15N solution to each pot of soil. Mix the soil every day for 3 weeks. During the balance process, use a weighing method to supplement soil water. After the L-glutamine-15N is supplied, take the soil sample, and store it at −20°C, which is recorded as D1.

Add L-glutamine-15N-treated soil into the plastic cup (the soil volume is about 80%), and press it as tightly as possible. The hole punch will punch a hole with a diameter of 2.5 cm in the middle. The rigors inoculated with a spore suspension concentration of 5 × 107 conidia/mL were placed into soil holes, the surface was covered with 1- to 2-cm-thick soil, the surface of the plastic cup was covered with a preservative film, the hole was tied with a toothpick, and it was cultivated in a dark place at 25°C for 1 month. Rinse the surface soil of mature C. chanhua, and then suck up the surface water with sterile paper. After cutting off the fruiting bodies, tear off the mycoderm, and collect the samples of C. chanhua mycoderm (D2) and inner sclerotia (D3) on the ultrapure clean workbench in a sterile centrifuge tube. Scrape the soil (D4) samples within 2 cm around C. chanhua into a ziplock bag. Figure S8 shows the experimental process and sampling diagram of 15N flowing from soil to insect body through C. chanhua mycoderm. Five treatments were mixed into one sample, three of which are repeated, and stored at −20°C. After the live tussah pupa is disinfected on the surface, it is covered with soil treated with isotopes. After the same treatment time as the experimental group, the tussah pupa is taken as the control group sample (Dck), and the 15N abundance value is compared with the treatment group.

15N flows from the insect body to the soil through the C. chanhua mycoderm (recorded as Experiment E)

Conidial suspension with a concentration of 5 × 107 conidia/mL was prepared, and 8 mg of L-glutamine-15N was added to each milliliter of spore suspension, which was completely dissolved and shaken well. Inject 0.02 mL conidia suspension + L-gluta­mine-15N solution into the third ring just behind the wing of each silkworm pupa, clip it into a sterile glass bottle with sterile tweezers, and keep it moist at 25°C for 5–7 days. After the silkworm pupa is infected and forms a stiff worm, take out the inner sclerotium sample for preservation, and record it as E1.

Put fresh soil into a disposable plastic cup with a soil volume of about 80%, and try to compress the soil. Punch a hole with a diameter of 2.5 cm in the middle of the plastic cup containing soil. Put the L-glutamine-15N-labeled zombie into the soil hole, and cover the surface with about 2 cm of soil. Cover the surface of the plastic cup with a fresh-keeping film, and tie a hole to keep moisture and breathable. Cultivate at 25°C in a dark place for 1 month. After C. chanhua matures, collect C. chanhua mycoderm (E2), inner sclerotium (E3), and soil (E4) samples within 2 cm around C. chanhua, and store them at −20°C.

Add 8 mg L-glutamine-15N to each 1 mL of sterile water, shake well after it is completely dissolved, inject 0.02 mL of this solution into each silkworm chrysalis, and cover it with soil at the same time as the experimental group. After the same treatment time as the experimental group, take soil samples (Eck) within 2 cm around the insect body to verify the transportation of 15N isotopes of the insect body to the soil in the presence of C. chanhua mycoderm. The experimental process and sampling diagram of the flow of 15N from the insect body to the soil through the C. chanhua mycoderm are shown in Fig. S9.

The test samples were sent to Jiangsu Nanjing Carvensys Testing Technology Co., Ltd. for determination of 15N content, with detection equipment SerCon Integra 2 Integrated EA-IRMS.

Total bacterial DNA extraction, PCR amplification, and high-throughput sequencing

E.Z.N.A. Soil DNA Kit is used to extract the total microbial DNA according to the operation procedure. The upstream and downstream primers 338F (5′-ACTCCTACGGGGAGGCAG CAG-3′) and 806R (5′-GACTACHVGGGTWTCTAA T-3′) were used for PCR  amplification. Use the PCR instrument (ABI Gene Amp 9700) for PCR reaction parameters: 95°C for 3 min, 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and 30 cycles. At 72°C, the reac­tion was prolonged for 10 min until the reaction stopped at 10°C (66). The  amplified products were sent to Illumina MiSeq sequencing platform of Shanghai Meiji Biomedical Technology Co., Ltd. for high-throughput sequencing.

Sequence processing and OTU comments

The PE reads obtained by MiSeq sequencing are spliced according to the overlap relationship, and the sequence quality is controlled and filtered at the same time. Then, according to the barcodes and primer sequences at both ends of the sequence, we can distinguish samples to get effective sequences and correct the sequence direction to get the optimized sequence. After all the original sequences were filtered and optimized and the chimeras were removed, OTU clustering was conducted on the QIIME platform and compared with the UNITE fungal database to obtain the taxonomic information of each OTU and make taxonomic analysis based on representative sequences (67).