Entomopathogenic Fungi in the Soils of China and Their Bioactivity against Striped Flea Beetles Phyllotreta striolata

The present research aims to explore the occurrence and diversity of entomopathogenic fungi (EPF) in cultivated and uncultivated lands from different provinces of China and to search for EPF against Phyllotreta striolata. In this study, first, the EPF biodiversity from the soil of four provinces (Hunan, Hubei, Henan and Hebei) was surveyed. There were 302 fungal isolates obtained from 226 soil samples collected from croplands (114), arbor (79), grasslands (97) and fallow land (12); 188 EPF isolates were identified as 11 genera. The data indicate that Hubei Province has the greatest EPF diversity, with a Shannon Evenness Index (SHEI) value of 0.88. Here, the grassland, arbor and cropland had an EPF diversity with SHEI values of 0.81, 0.86 and 0.76, respectively, while the fallow land had the highest SHEI value of 1.00, which suggests that cultivation by humans affected the count and richness of soil fungi: the less human activity, the more kinds of fungi found. Finally, the pathogenicity of 47 fungal strains against the adult P. striolata was determined. Isaria javanica (IsjaHN3002) had the highest mortality. In conclusion, this study reports the EPF distribution and biodiversity in the soil from four provinces in China, showing that the amount and type of fungi in the soil varied by region and vegetation and that soil was one of the resources for acquiring EPF. The potential of I. javanica as a biocontrol must be studied further.


Introduction
Entomopathogenic fungi (EPFs) are ubiquitous in nature. Biological plant protection with EPFs plays a key role in sustainable pest management programs [1]. In addition to absorbing nutrients for their own growth, some EPFs can control insect populations at low levels for long periods [2]. Fungi-based insecticides have great potential as a form of pest control [3]. Not only are EPFs are harmless to human beings, animals and crops, but they also have the advantages of long-term validity, non-resistance, no residue, no pollution, no damage to natural enemies, high epidemic potential and ease of production [4,5]. Therefore, using EPFs to control agricultural and forestry pests has become a new trend in pest control. EPFs are the largest group of insect-pathogenic microorganisms. According to incomplete statistics, about 100 genera and 1000 species of EPFs have been recorded around the world [6], and more than 40 genera and more than 400 species have been found in China [7], including Beauveria, Metarhizium, Penicillium and Fusarium. Beauveria bassiana and Metarhizium anisopliae have been extensively developed as mycoinsecticides [8]. These species are naturally present in agricultural soils, but the spore numbers in nature are often too low to result in the effective control of pest population outbreaks [9].
Through in-depth studies on the physiology, ecology and molecular biology of EPFs, the effect of applying EPFs to control insects has been significantly improved. Under the premise that pests generally develop resistance, more and more attention has been paid to sustainable development and pollution-free pest management, and researchers

Soil Sample Collection
The soil samples were collected from different sites (cropland, fallow land, arbor and grassland). The longitude and latitude of each site were recorded by ICEGPS 100C (Shenzhen, China). From each site, approximately 200 g of soil (10~15 cm depth) from three points was collected, mixed and stored in a plastic bag at 4 • C until further use. In total, 226 samples were collected from these sites (Table A1, Appendix A).

Isolation of Fungi from the Soil Samples
The method from our previous work was used to isolate fungal strains from the soil samples [27]. Soil suspensions of 0.02 g/mL were prepared with 0.1% Tween-80 solution; then, 0.1 mL of the suspension was inoculated onto a selective medium (PDA, 0.2 g/L cycloheximide, 0.2 g/L chloramphenicol and 0.013 g/L Bengal red) and cultured at 25 ± 1 • C. When the fungi grew out, a single colony was transferred onto the PDA plate and cultured at 25 ± 1 • C, purified and cultured until a new colony was formed [28].

Identification of Fungal Species and Analysis of Genetic Homology
The identification of fungal isolates was based on the morphological characteristics and similarity of the rDNA-ITS sequences. DNA extraction kits (DP3112, Bio-Teke, Beijing) were used to extract the total DNA from fungal isolates. The primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) were used to amplify the ITS region on a T100 TM Thermal Cycler (BIO-RAD, Hercules, CA, USA) via a standard PCR cycling protocol (94 • C for 3 min, 94 • C for 30 s, 55 • C for 30 s and 72 • C for 1 min for 33 cycles, then 72 • C for 10 min). The obtained ITS rDNA sequences were submitted to GenBank and compared with similar sequences through the BLAST tool of NCBI. The phylogenetic trees of the fungi were constructed by MEGA X via the statistical method of maximum likelihood, a bootstrap test of 500 replications and the Jukes-Cantor model [29]. The fungal strains are listed in Table 1.

Evaluation of the Shannon Evenness Index
The biodiversity of fungi and EPFs in different soils was evaluated using the Shannon Evenness Index (SHEI). The SHEI was calculated via the formula SHEI = −∑ s i (Pi)(ln Pi)/lnS, where s is the total number of species in the sample, i is the total number of individuals in one species, Pi is the proportion of species in the sample, lnPi is the value of the natural logarithm of Pi and S is the total number of species.

Bioassay of the Fungal Strains against P. striolata
The isolates of fungal species were subject to a bioassay against P. striolata based on the work of [27]. In summary, fungal conidia suspensions of 1.0 × 10 8 spores/mL were prepared with 0.02% Tween-80 solution. Spore suspension concentrations of 1.0 × 10 4 , 1.0 × 10 5 , 1.0 × 10 6 , 1.0 × 10 7 and 1.0 × 10 8 spores/mL were prepared by culturing with a light cycle of 12:12 at 25 • C for 7 days. The population of P. striolata was fed with radish lumps, which changed every day. Adults were paralyzed with carbon dioxide and dipped into the conidial suspension for 20 s. The pest populations were surveyed every 24 h after treatment. The 0.02% Tween-80 solution was used as a control group. The experiment was replicated thrice, and 20 adults were used for each treatment.

Scanning Electron Microscopy
The samples were placed in a 2 mL centrifuge tube, fixed with 2.5% glutaraldehyde overnight, washed with physiological saline and dehydrated using a graded series of ethanol; isoamyl acetate was replaced overnight. They were vacuum-dried, fixed onto the platform and then coated with platinum with an ion coater before being observed using a scanning electron microscope.

Statistical Analysis
Analyses of the bioassay data were carried out using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA). The data were expressed as mean ± SD and were subjected to one-way ANOVA, followed by Duncan's multiple range test (DMRT). Significant differences were accepted at p < 0.05.

Distribution of Soil EPF in Different Regions
There were different numbers and isolating rates of EPFs in different regions. Compared with the average fungal isolating rates of 83.70% and 61.92% in all fungi and EPFs, Henan had the highest rate of >90% (Table 2). However, the Shannon Evenness Index indicated that Hubei and Hunan were districts with the highest EPF biodiversity, while Hunan and Hebei had the EPF biodiversity with SHEI values of 0.87 and 0.88, respectively ( Table 2).

The Biodiversity of Soil EPF in Different Environments
There were different numbers and isolating rates of EPF in Central China. Compared with the average fungal isolating rates of 87.42% and 61.16% for all fungi and EPFs, cropland samples had higher rates of >69% (Table 3). However, the SHEI indicated that cropland had the lowest EPF biodiversity, while fallow land samples had the most abundant EPF biodiversity (Table 3).

The Pathogenicity of Fungal Isolates against P. striolata
Forty-seven isolates were subjected to a bioassay against P. striolata. The results indicate that I. javanica (IsjaHN3002) had the highest mortality, and Aspergillus spp., Fusarium falciforme, Lecanicillium spp., Metarhizium spp. and Talaromyces spp. all had obvious pathogenicity against P. striolata (Table 4).

The Pathogenicity of I. javanica against P. striolata
According to the results shown in Table 5, the number of muscardine cadavers increased with the spore concentration. The lethal rate of 1.0 × 10 8 spores/mL spore suspension treatment group was as high as 80%. When the spore concentration was lower than 1.0 × 10 6 spores/mL, no hyphae were observed on the body wall of P. striolata in the first 3 days. There was no significant difference in the rate of zombies in the groups treated with spore suspensions at concentrations of 1.0 × 10 4 and 1.0 × 10 5 , 1.0 × 10 6 spores/mL in the first 3 days, but there was a significant difference in the rate of zombies in the group treated with spore suspensions with concentrations of 1.0 × 10 7 and 1.0 × 10 8 spores/mL in the first 3 days. After the seventh day, the differences among the treatment groups were revealed. Compared with other treatment groups, there was a significant difference in the lethal rate of the spore suspension with a concentration of 1.0 × 10 8 spores/mL.

Scanning Electron Microscopy Observations of Infection Process of I. javanica
The results showed that the attachment of conidia of I. javanica to different parts of the body surface was very different. After 2 h, the attachment of conidia was observed. No attachment of conidia was found on the head, abdomen, shard or other smooth surfaces. The conidia were mainly attached to the bristly areas and internodes such as the antennae, foot joints, chest and chest feet. The most densely attached site was the intersegmental membrane of the chest feet, followed by the foot joints ( Figure 6). After 12 h of inoculation, some conidia began to germinate, forming short germ tubes at the top. Twenty-four hours after infection, the top of the germ tube expanded to form an appressorium and continued in the direction of the intersegmental membrane, forming tendrils ( Figure 7A-C) and looking for a suitable invasion site. The germ tube could also directly invade the body wall ( Figure 7D). At 48 h, hyphae began to grow between the foot internode, and new conidiophores and conidia sprouted ( Figure 8A). Next, 48-72 h after inoculation, the surface of the insect body was gradually covered by mycelia until it was completely covered ( Figure 8B-D). Through stereoscopic observation, the mycelia were observed to grow from the body surface on the third day, and then the mycelium coverage increased day by day (Figure 9), while the control group never experienced mycelial growth.

Discussion
This study surveyed the EPF distribution at a broad scale in China. ITS sequences are small and easy to analyze and have been widely used in the phylogenetic analysis of different fungal species, but their accuracy is controversial. Therefore, the identification of the fungal species in this study has some defects. Undoubtedly, our results initially provide a large amount of information about the soil fungi in these areas. Moreover, the results indicate that the soil environment strongly impacts the distribution of EPFs. Compared to arbor and non-cultivated land, the cropland samples had fewer EPFs. The isolation rate of EPFs was not high, which showed that soil fungi were not abundant in these areas and that the sampling and isolation methods also affected the isolation of fungi. The EPF diversity may be affected by the use of fungicides in croplands. China is a heavy consumer of pesticides, and a large number of broad-spectrum fungicides such as carbendazim, chlorothalonil and azoxystrobin, etc., are sprayed on croplands and probably inhibit fungi [59,60].
EPFs can parasitize insects and cause insect diseases, including some obligate parasitism that may not cause insect death but that can reduce the vitality of the host insects and weaken them [61] or affect insect spawning [62]; as such, when using EPFs, we can observe changes in the behavior of host insects [63,64]. Some studies have suggested that insects can actively identify fungi, with the target location being the cell wall of the fungi, while the fungi will take a series of measures to evade the host's defenses in the face of insect recognition [65]. Therefore, the invasion of host insects by EPF is a process of mutual influence and interaction [66]. As a result, the body surface of P. striolata may be able to recognize I. javanica, and the resistance and defense of I. javanica may also take measures to promote the germination of conidia in advance. In view of this fact, we can further explore what receptor binds the cell wall of conidia of I. javanica to produce signal molecules and promote spore germination, determining the factors promoting spore germination and improving pathogenicity.
Through scanning electron microscope observation, 12 h after infection with I. javanica, some conidia began to germinate, as shown in Figure 7. After 24 h of infection, only some scattered spores germinated. Because of the hard shell and dense structure on the body surface, the structure of the body wall varies greatly in different parts, and the outer skin has hydrophobic components. However, in tests of the bioactivity of different concentrations of spore suspensions against P. striolata, it was found that the spore suspension concentration of I. javanica had a stimulative effect on the production of zombies. This may be the QS phenomenon observed in I. javanica, which refers to a change in the physiological and biochemical characteristics of the microbial population in the process of its growth due to an increase in the population density, showing the characteristics of a small number of bacteria or a single bacterium. Cells use the QS mechanism to carry out cell-to-cell communication so that they can coordinate in a complex environment, and their "team combat ability" better ensures that the whole population survives. At present, the study of QSM is mainly focused on bacteria, and QSM has also been reported in related fungi [67]. In recent years, more reports have confirmed that fungi have QSM [68,69] and have QSM pheromones that are similar to the bacterial regulation of the physiological behavior of fungi [70][71][72]. However, in-depth studies of fungal QSM have not been carried out. Therefore, in the production of fungicidal insecticides using I. javanica, we can choose the appropriate formulation or use new production technology to help I. javanica survive in the form of sporangia, and it can also attach to the body surface after application to invade the body faster and improve its pathogenicity.
Several species have not been reported as EPF, namely Aspergillus, Lecanicillium, Monascus, Talaromyces and Fusarium. Their pathogenicity against P. striolata was discovered, and their potential for pest control deserves further research. Our experiment will provide new insight into the distribution characteristics of EPF and the conservation of their biodiversity.

Conclusions
In conclusion, 188 EPF isolates were identified from 226 soil samples, and the amount and types of fungi in the soil varied by region and vegetation type. Metarhizium, with 89 isolates, was recognized as the dominant EPF species, whereas Purpureocillium and Beauveria (respectively with 81 and 11 isolates) were the richer genera. Finally, it was first reported that I. javanica had pathogenicity against P. striolata, and we described its infection process.

Conflicts of Interest:
The authors declare no conflict of interest.