Distribution and pathogenicity of Beauveria bassiana in soil with earthworm action and feeding.

Earthworm action and feeding have an important impact on a variety of microorganisms in the soil. However, the effects of the earthworm on Beauveria bassiana, a common entomopathogenic fungus in the biological control of pests, have been little studied. In this study, the epigeic earthworm species Eisenia fetida (Savigny) was selected to evaluate its impact on B. bassiana TST05 including its distribution in soil and its pathogenicity to target insects. By testing B. bassiana TST05 distribution, biomass in soil, viable spore germination rate, and pathogenicity to insect larvae after passing through the earthworm gut, the results showed that the activity and feeding of E. fetida promoted the B. bassiana TST05 diffusing downwards in the soil, while decreasing active fungal spores. After passing through the earthworm gut and excretion, the living B. bassiana individuals still had activity and pathogenicity to insects. The germination rate of the viable fungal spores was 15.09% and the infection rate to the insect larvae of Atrijuglans hetaohei Yang reached 62.35%, 80.95% and 100% after infection at 7 d, 10 d, and 14 d, respectively. The results showed that action and feeding of earthworms promoted the distribution of B. bassiana TST05 in soil, but decreased B. bassiana viable spores. This study is important for understanding the interaction between earthworms and B. bassiana in soil and for guiding the scientific application of B. bassiana in the biological control of pests.

1. Introduction

Earthworms, belonging to the order Opisthopora and class Oligochaeta in the phylum Annelida, are the soil organisms with the largest biomass in temperate terrestrial ecosystems and play important ecological functions. Earthworms have positive effects on soil aeration, nutrient cycling, soil structure and fertility, plant growth and organic matter accumulation and transformation by their feeding, excretion and movement [1, 2]. In the process of feeding, earthworms devour a large amount of soil, among which microorganisms in the soil are an important food and the source of essential amino acids for earthworms [3]. Earthworm feeding often leads to the reduction of some microorganisms and the proliferation of others [4]. It was reported that earthworms might serve as phoretic hosts to entomopathogenic nematodes and B. bassiana. However, it was difficult for entomopathogenic nematodes to survive after they passed through the gut of earthworms, and the transmission of entomopathogenic nematodes by earthworms mainly depended on soil churning and mixing [5-8]. However, there is no report on the important questions, i.e., Whether B. bassiana can survive when passing through the gut of earthworms, and whether earthworms can spread B. bassiana by excreting earthworm casts.

Beauveria bassiana is one of the most common entomopathogenic fungi in the world [9], and has a wide host insect range from multiple orders [10]. In agricultural production, B. bassiana has been widely used as a biopesticide in farmlands, orchards, vegetable fields and forests [11]. It has been noted that the number of B. bassiana applied into soil can vary greatly with different times, places and treatment methods [12], and soil temperature and humidity are important factors [13-16]. Quintela et al. (1992) suggested that B. bassiana in soils with high humidity can be decreased by some competing microorganisms or other inhibiting organisms [14]. However, little attention has been given to the impact of earthworms, one of the largest biomass animals in the soil, on B. bassiana [1].

In this study, the epigeic earthworm species E. fetida (Savigny) (Opisthopora: Lumbricidae), B. bassiana TST05 strain (Moniliaceae: Beauveria), and an insect A. hetaohei (Lepidoptera: Heliodinidae) were selected to investigate the effects of earthworm action and feeding on the distribution of B. bassiana and its pathogenicity to target insects.

The insect A. hetaohei is an important pest of walnut fruit in northern China. The mature larvae of A. hetaohei coexist with earthworms and B. bassiana in the same soil environment for 8–9 months. Under natural conditions, larvae are often infected by entomogenous fungi such as B. bassiana in soil. The biological control of A. hetaohei using B. bassiana and other entomopathogenic fungi has attracted much attention [17]. It was reported that a high infection mortality of mature larvae was achieved using B. bassiana strain TST05 to infect A. hetaohei which was applied in walnut orchard soil [18]. Therefore, the mature larvae of A. hetaohei were selected as the target insects infected by B. bassiana strain TST05 as the experimental material in this study.

Beauveria bassiana TST05 strain is a highly pathogenic strain that was originally isolated in 2009 by our laboratory from the naturally infected overwintering larvae of Carposina sasakii (Matsumura) (Lepidoptera: Carposinidae) in the soil of apple orchards in Xiangfen County, Shanxi Province, China. The strain was identified as B. bassiana by molecular technology [19]. The results of sequence alignment were consistent with those of morphological identification. Therefore, the TST05 strain was identified as B. bassiana. The biological characteristics of the B. bassiana TST05 strain, pathogenicity to host insects, persistence in soil, and compatibility with chemical insecticides were studied in our laboratory [20-22]. At the same time, the strain was deposited in the China General Microbial Species Conservation and Management Center (Beijing, China) under storage number CGMCC4526.

The aim of the study was to learn how to influence the diffusion of B. bassiana in the soil by the earthworm action behavior, how to influence quantity and vitality of B. bassiana by earthworm feeding and digestion, and whether the viable B. bassiana spores in the casts of the earthworms still keep infectivity to the target insects, in order to further understand whether the activities of earthworms in the soil will have an impact on the number, distribution and pathogenicity of B. bassiana, and provide a reference for the impact of earthworms on biological control and the application of B. bassiana as a biological pesticide.

2. Materials and methods

2.1 Earthworm, fungus and insects

Earthworm E. fetida samples were obtained from the earthworm breeding base in Baiyangdian, Hebei, China, and raised in the animal culture laboratory of the School of Life Science, Shanxi University. Laboratory conditions were 20°C in temperature with 70% relative humidity (RH). The earthworms were maintained in sterilized soil with 40 cm soil thickness and 16% soil moisture. The soil used in the experiment was collected from a forest and was composed of sandy soil 52%, silt 31%, and clay 17%. Fresh fruits and vegetables were put into the soil regularly as a feed for earthworms. Adult clitellate earthworms of similar body size (weight 0.3–0.5 g and length 5.0–5.5 cm) were selected for this study. The earthworms were washed with sterile water for several times until there was no sediment on the surface of the earthworm, then placed in a Petri dish that was covered with wet filter paper and fasted for 48 h to avoid any cross contamination with their casts before they were treated.

The strain used in this test was the isolated and identified strain B. basiana TST05 from our previous study [19]. It was inoculated on Potato Dextrose Agar (PDA) medium, and cultivated in the laboratory with 25°C, 75% RH and a photoperiod of 16:8 h (L: D); fungal conidia were collected by 5 d for the study [20].

The mature larvae (in four or five instar stages) of A. hetaohei (Lepidoptera: Heliodinidae) were selected as the target insects infected by B. bassiana strain TST05. Before the experiment, walnut worm fruit was collected in late August from an experimental walnut orchard in Yuxian County, Shanxi Province, China, and then placed in the laboratory with 50% ~ 70% RH (Longitude 112.58754, latitude 37.79975). When the mature larvae naturally drilled out of the fruit, they were collected and transferred in a culture dish covered with a wet filter for fungal infection study.

2.2 Test of the diffusion effect of earthworms on B. bassiana spores in soil

The soil column followed the method of Shapiro-Ilan & Brown (2013) [8]. PVC pipes with a diameter of 17cm and a height of 20cm were divided into four equal parts with each part 5cm high, and the four equal parts of PVC pipes were glued together with adhesive tape. The motion range of the earthworm E. fetida was confined to the soil column that was stacked with four-section polystyrene plastic tubes. The plastic tubes were fully filled with 7000 g of sterilized soil with moisture content of 16%.

A spore suspension of B. bassiana strain TST05 was prepared at 2.5×108 spores/mL, and 25 mL of the suspension sprayed on the surface soil in each column. Fifty adult individuals of E. fetida with a biomass of about 20g were placed on the soil surface of each column and kept for four hours at room temperature. Then, the soil columns were maintained at 25°C with 75% RH and a photoperiod of 16:8 h (L: D) for 7 days. The moisture was maintained by adding 12 mL of water every other day. Negative control (CK) groups without earthworms were used for comparison. Three replicates for each treatment and control were performed.

After 7 days, about 1750g soil from each section was transferred to a sterile glass tank and completely mixed by stirring. The soil sample was collected from each section tube and placed into Petri dishes. The soil block was crushed and kept for 15min. Then, 1g dry soil was weighed and diluted 10,000-fold with sterile water.

Then, one mL of the mixture was inoculated on PDA medium and cultured at 25°C and 75% RH with a photoperiod of 16:8 h (L: D). After culturing for 7 days, the fungal colonies were observed and counted referred Xiong’s method [19]. Each group was treated for five repeats.

2.3 Detection of B. bassiana biomass in the earthworm gut and cast

A total of 4 kg dry sterilized soil was inoculated with 640 mL spore suspension (2.5×106 spores/mL) of B. bassiana strain. A total of 250 individuals of E. fetida with a biomass of about 100g were transferred into the inoculated soil and maintained at 25°C for 3 days. Negative control (CK) groups were performed without earthworms. After 3 days, in the treatment group, 200 individual earthworms were removed, washed and dissected. The contents of the foregut and midgut of the earthworms were collected. The remaining 50 earthworms were washed, and put into sterile Petri dishes lined with wet filter paper at a shaded place for their casts to be collected. Three replicates were done for both treatments and controls.

The foregut contents, midgut contents and casts of the earthworm E. fetida from the treatment group were air-dried in sterile Petri dishes, and the cast granules were crushed into powder by gently pressing them with a sterilized small spoon. One-gram dry samples of the soil as well as the foregut contents, midgut contents and casts were diluted 10,000-fold with sterile water into the mixture. Then, these mixtures were inoculated on PDA medium; the inoculated amount was a 1-mL mixture for each plate. After inoculation, these plate PDA media were cultured at 25°C and 75% RH for 7 days, and their fungal colonies were observed and counted [19]. Each group was repeated for five times.

The soil samples in the control group (CK) without earthworms were collected and the biomass of B. bassiana TST05 was detected using the same method as used in the detection of the foregut contents, midgut contents and casts of the earthworm E. fetida. The experiment was repeated for five times.

2.4 The influence of earthworm digestion on the pathogenicity of B. Bassiana

A total of 3kg dry sterilized soil was put into a glass tank (25cm×25cm×40cm), and the thickness of the soil was 20 cm. We inoculated 480 mL of a 5×109 spores/mL spore suspension of B. bassiana TST05 into the sterilized soil with the number of B. bassiana spores for 3.84×1013 spores/m2 in a 0-20cm soil layer. It has been reported that the application amount of B. bassiana in soil is about 1012~1014 spores/m2 in a 0-20cm soil layer [11, 23, 24]. The number of B. bassiana spores in the experiments was consistent with the amount of B. bassiana TST05 powder used in field experiments. It has been reported that the biomass of earthworms is 1.60–530.12 g/m2 in a 0-20cm soil layer in farmland [25]. Here, 80 individuals of the earthworm E. fetida with a biomass of 33 g were introduced into the inoculated soil, and the biomass of earthworms was 528 g / m2, which was consistent with earthworm biomass in farmland soil. After being transferred into the soil, the earthworms were maintained at 25°C for 3 days. Then, 80 individual earthworms in the treatment group were removed, washed, and put into sterile Petri dishes lined with wet filter paper, and their casts were collected in a shaded space. The experiment was repeated for three times.

In the control group (CK), B. bassiana was inoculated but without earthworms, and soil samples were collected after three days. The experiment was repeated for three times. Two grams of the dry cast of E. fetida was mixed into 20 mL of sterile water and settled for 10 min. Then, the supernatant was prepared into a spore suspension at a concentration of 1×107 spores/mL. The larvae of A. hetaohei were inoculated by soaking in the mixture for 5 s and then transferred in a Petri dish lined with wet filter paper and absorbent cotton at the bottom. Forty larvae in each Petri dish were put in a 25°C incubator to be observed every day for their infection symptoms. There were three replicates for the treatment group. The dead larvae were counted and calculated for their corrected mortality. In the control group (CK), the soil samples were used instead of the earthworm casts. There were three replicates for the control group. To calculate corrected mortality, the larvae of A. hetaohei were soaked in sterile water instead of the mixture. Three replicates were performed.

2.5 Statistical analyses

Prior to statistical analysis, all variables expressed as frequencies were arcsine transformed, while quantitative variables were log (x +1) transformed. Data were analyzed using the SPSS 21.0 statistical software package. ANOVA and Tukey’s HSD were performed for statistical analyses. Statistical differences were assessed for P < 0.05.

3. Results

3.1 The effect of earthworms on the diffusion of B. bassiana in soil

The earthworm E. fetida crawled and drilled quickly into the soil once placed on the surface of the soil column (Fig 1A). When the soil columns were taken apart after 7 days, the earthworm distribution in different soil layers was observed, and approximately 80% of earthworms were distributed in the second and third layers of the soil column.

Fig 1

Effect of earthworm presence on dispersal of B. bassiana.

(A): Soil column on dispersal of B. bassiana in the presence of earthworm E. fetida; (B): Colony culture of B. bassiana; (C): Statistical analyses of the colony number of B. bassiana in each layer of soil column. CK: control group with B. bassiana only; EW: treatment group included both B. bassiana and E. fetida. 1–4 represent the first layer (0-5cm), the second layer (6-10cm), the third layer (11-15cm) and the fourth layer (16-20cm) of soil column, respectively.

In the treatment group with earthworms (EW), the colonies of B. bassiana in the first and second soil layers had the largest numbers (Fig 1B, EW) at 16–21 CFU g-1 and 16–19 CFU g-1, respectively. Fungal colony numbers were not significantly different between the first and second layers of soil. While, the fungal colonies in the third layer significantly decreased to 10–15 CFU g-1. The fourth layer soil contained the least fungal colonies at 1–3 CFU g-1, which was significantly different from the other three layers (Fig 1C).

In the control group without earthworms (CK), a large number of B. bassiana colonies, 180–260 CFU g-1, were isolated from the first layer of the soil column (Fig 1B, CK). However, the second layer soil contained only 0–2 CFU g-1 B. bassiana colonies, and no fungal colonies were observed in the third and fourth layers of the soil column (Fig 1C). In the control group without earthworms (CK), after 7 days of incubation, the total number of B. bassiana spores in the soil column was approximately 3.85×109, but in the treatment group with earthworms, it decreased to 8.4×108. The difference between them was significant.

3.2 Effect of earthworm digestion on spore germination of B. bassiana

The colony of B. bassiana in each sample was mounted by collecting samples from the soil in the control group (CK) and from the foregut contents, midgut contents and casts of the earthworm E. fetida in the treatment group; by culturing these samples on plate medium, the colony numbers of B. bassiana in each sample were determined. The average numbers of colonies isolated from the soil, foregut, midgut and cast samples were 21.2, 19.4, 11.8 and 3.2, respectively (Fig 2). The soil sample contained the most B. bassiana, while the cast sample had the fewest. The B. bassiana spores were swallowed with the soil and entered the digestive tract of the earthworm. No significant difference between the B. bassiana colonies isolated from the foregut contents and the soil sample, which showed that when they first entered the digestive tract, B. bassiana spores were less affected in the foregut, and 91.51% were still alive. The colonies isolated from the midgut contents of the earthworms were fewer in number, indicating that great damage occurred to B. bassiana and resulted in a survival rate of 55.66%. After digestion in the midgut of the earthworm, the remaining spores of B. bassiana passed through the hindgut and were excreted in the cast where the survival ratio of the spores was only 15.09%.

Fig 2

Effect of earthworm feeding on spore germination of B. bassiana.

(A): The colony culture of B. bassiana in the soil, and in the digestive tract and cast of E. fetida; (B): Statistical analyses of the colony numbers of B. bassiana. CK represents in soil, Fg in the content of earthworm foregut, Mg in the content of earthworm midgut, and Ca in the content of earthworm casts, respectively.

3.3 Pathogenicity of B. bassiana in the casts

The infection experiment was conducted on the larvae of A. hetaohei, a pest on walnuts, to test the pathogenicity of B. bassiana surviving in the casts of E. fetida. Either the larvae inoculated with B. bassiana in the soil sample (CK group) or these the larvae inoculated with B. bassiana that survived in the cast of E. fetida (treatment group) showed the similar infection symptoms, disease courses, and mortality. After infection for two days, the vitality of the larvae decreased. At three days, the body color of the larvae changed to yellow and dark, some larvae began to die, and their bodies gradually became shrunken and shriveled (Fig 3A1 and 3B1). At five days, villous hyphae appeared on the cuticle of the larvae (Fig 3A2 and 3B2). At eight days, the dead larvae became stiff and were partly covered with white hyphae (Fig 3A3, 3B3). At 12 days, the hyphae became thick and completely covered the dead larval bodies (Fig 3A4 and 3B4). At 14 days, dense spores appeared on the body surfaces of the dead larvae (Fig 3A5 and 3B5).

Fig 3

Disease symptoms of show the infection symptoms of larvae being inoculated with B. bassiana in soil on the 3rd, 5th, 7th, 10th and 14th days, respectively. (B1~B5) show the infection symptoms of larvae being inoculated with the B. bassiana in the casts on the 3rd, 5th, 7th, 10th and 14th days, respectively.

The larval mortality rate at 14 days showed a gradual increase. After three days, the cumulative corrected mortality of the larvae in the treatment group infected by B. bassiana that survived in casts of E. fetida was 14.61±5.76%, while that in the CK group infected by B. bassiana that survived in soil was 15.73±7.64%. At fifth day, the cumulative corrected mortality rates were 34.48±6.03% in the treatment group and 48.28±3.00% in the CK group. At seventh day, the cumulative corrected mortality rates were 62.35±3.68% in the treatment group and 75.29± 5.85% in the CK group. At tenth day, the cumulative corrected mortality rates were 80.95±4.17% in the treatment group and 86.90±3.10% in the CK group. At 14 d, the cumulative corrected mortality was 100% in the treatment group and 100% in the CK group (Fig 4). The mortality rate of the larvae in the treatment group was slightly lower than that in the CK group, but the difference was not significant. The median lethal time of the larvae in the treatment group was 6.503 d, while it was 5.583 d in the CK group. Although infection and death of the larvae required more time in the treatment group than in the CK group, the difference between the two groups was not significant (Fig 4). This showed that B. bassiana that survived in casts of E. fetida still had high infectivity to A. hetaohei larvae.

Fig 4

Corrected mortality of A. hetaohei larvae after being inoculated with the B. bassiana in the cast of E. fetida.

CK: control group of B. bassiana in soil; Ca: treatment group of B. bassiana in the casts.

4. Discussion

Beauveria bassiana is one of the most important entomopathogenic fungi in the world, and the soil is its main habitat [12, 14]. Connections and interactions between B. bassiana and other organisms in soil affect the survival, vitality and infectivity of the fungus [10]. Among the numerous soil organisms, earthworms represent the largest animal biomass in soil [1]. It is known that earthworm activities have an important impact on the physiological metabolism and population ecology of some microorganisms in the soil [26-28]. In this study, the earthworm E. fetida was used as an example to investigate the effects of earthworm action and feeding on the distribution and pathogenicity of B. bassiana TST05 in soil.

Four layer soil columns were employed to assess the effect of E. fetida activity on the distribution of B. bassiana TST05 in the soil. The results showed that downward diffusion of B. bassiana TST05 spores was not obvious in the control group without the presence of the earthworms. The vast majority of B. bassiana TST05 spores were still concentrated in the first layer of the soil column. In comparison, the distribution of B. bassiana TST05 spores in the soil column changed significantly in the treatment group in the presence of earthworms. Beauveria bassiana TST05 colonies were isolated from all four layers of soil samples in the column. This was due to earthworm action to promote B. bassiana TST05 spores diffusing downwards, which resulted in the decrease of B. bassiana spores in the first layer of the soil column, and the increase in the second, third and fourth layers. The current result is essentially consistent with that of Shapiro-Ilan and Brown (2013) [8], who used the earthworm species L. terrestris L., while the earthworm E. fetida was used in our study. However, the activities of these two earthworms both promoted the downward diffusion of B. bassiana inoculated on the surface of the soil column. In addition, the results also showed that the total number of B. bassiana colonies isolated from soil in the treatment group with the presence of earthworms decreased significantly by 78% compared with the control group. It indicated that earthworm activities may promote the downward diffusion of B. bassiana in soil but it reduced the number of active spores of B. bassiana [10, 29].

The earthworms promote B. bassiana diffusion in soil by two methods: one is to attach and carry spores by their body surface, and the second is to feed and excret through their digestive tract. Therefore, it is necessary to study the attachment of B. bassiana on the earthworm surface and part of earthworm epidermal mucus.

In the process of earthworm activities, they will devour a large amount of soil so that the fungi in the soil could enter the earthworms’ digestive tract, which may be one of the reasons for the decrease in the B. bassiana population. It has been found that due to the digestion of earthworms, the composition of fungal populations changed in their gut. The spores of some fungi survive in the midgut environment and begin to germinate and grow more actively in the fresh cast of earthworms. The specific environment of the earthworm gut may comprise the special "filters" and "fermenters" of some soil bacteria and fungi [30]. Moreover, there is no taxonomic relationship in the effects of earthworm digestion on soil bacteria and fungi. Sensitive and resistant populations can be found in the same genus of bacteria or fungi [31]. To observe the effect on the activity of B. bassiana after passing through the earthworm gut, E. fetida was fed in soil that was inoculated with B. bassiana spores, and then soil samples in the control group (CK) and the foregut contents, midgut contents and cast of E. fetida were cultured. Our results have shown that some B. bassiana can survive after passing through the digestive tract of earthworms, which can be carried and diffused with earthworm activities and excretion in the soil. However, at the same time, it was found that the total number of active B. bassiana spores decreased significantly during this process. Significant changes occurred in the midgut and the casts, and the number of B. bassiana spores detected from the midgut content samples was nearly half that of the soil samples; the spores detected in earthworm casts were only 15.09% of the soil samples. This may be due to the digestion of some B. bassiana spores by earthworms after passing through the digestive tract of earthworms.

Shapiro-Ilan and Brown (2013) confirmed that B. bassiana spores distributed in the soil column still had a high infection rate on Galleria mellonella (L.) larvae with the presence of L. terrestris L. [8]. In this study, to verify the influence of intestinal digestion of earthworms on the pathogenicity of B. bassiana TST05, the walnut fruit bore A. hetaohei was selected as the target insect for the infection test. The results showed that the infection processes and symptoms of the two groups were similar, and there were no significant differences in the lethal rate and median lethal time between the two groups. Xiong et al. (2012) used the same concentration of B. bassiana TST05 as in the current study to infect the larvae of Carposina sasakii Matsumura, and found that the median lethal time was 6.503 days [32], similar to our study. It is inferred that although the number of B. bassiana spores is significantly reduced through the earthworm intestinal digestion, the living spores of B. bassiana still have strong infectivity to the target insects.

This model takes advantage of the biological and ecological characteristics that A. hetaohei larvae fall into the soil after they mature and spend the winter in the soil from September to following May so that the insects are in the same soil environment as earthworms and B. bassiana.

5. Conclusions

E. fetida promoted the diffusion of B. bassiana in soil but significantly reduced the total amount of B. bassiana in the soil. In the process of earthworm feeding, B. bassiana enters the digestive tract together with the soil. After digestion, the surviving spores are excreted with the casts, which is an important way to promote the diffusion of B. bassiana and a process of reducing its quantity. After entering the digestive tract of earthworms, the survival rates of B. bassiana were approximately 55.66% in the midgut and 15.09% in the casts. However, living B. bassiana in the casts still had strong vitality and infection activity, which was manifest in the infection symptoms and mortality rate to the A. hetaohei larvae between the treatment and control groups. This is very important for B. bassiana in the biological control of pests.

Although this study provides evidence for the effects of earthworm activities, especially feeding and digestion, on the population and infection activity of B. bassiana in soil, it is necessary to further study the mechanism of earthworm digestion on B. bassiana. The earthworm epidermal mucus is an important immune barrier against microbial invasion. Therefore, it will be important to study whether and how earthworm epidermal mucus inhibits B. bassiana.

10 Feb 2022

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Reviewer #1: This study assessed the impact of the earthworm Eisenia fetida affecting on Beauveria bassiana, including its distribution in soil and pathogenicity to Atrijuglans hetaohei Yang. The result showed that action and feeding of earthworm may promote distribution of B. bassiana TST05 in soil, but decreased the viable spores of B. bassiana. The viable spores of B. bassiana TST05 passed through earthworm gut still had certain germination ability and higher pathogenicity to the insects. This study is important for guiding the scientific application of B. bassiana in biological control of pests. However, several questions should be addressed.

1.Beauveria bassiana TST05 strain that have been identified in the previous studies in line 67-79 do not need to be described in detail in Result section.

2.The statistical difference of fungal colony numbers between layers should been marked in Fig. 1 C and the statistical analyses between the CK group and the treatment group should been labelled in Fig. 5.

3. The discussion section need to be streamlined and logic, e.g. the content same as the result section be deleted and reasonable discussion.

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10 Mar 2022

Dear Editor,

Thank you and the reviewers very much for reviewing our manuscript and giving us valuable comments and suggestions.

According to your advice, we revised all the relevant parts in the manuscript, and responded point by point to the comments as listed below. We highlighted the changes in the revised manuscript by tracking in Word. We also did some edits for the mistakes.

We resubmit here the revised manuscript and hope this revision will be suitable for publication in PLOS ONE.

If you have any questions regarding the manuscript, please feel free to contact me.

With kindest regards,

Yours Sincerely,

Yingping Xie

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Response: The article has been modified according to the format and style requirements of PLOS ONE.

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Response: Our colleague Professor James Crabbe has checked the whole MS and re-written the text where appropriate, and so we have invited him as our co-author for his important contribution.

3. Beauveria bassiana TST05 strain that have been identified in the previous studies in line 67-79 do not need to be described in detail in Result section, L205.

Response: Thanks. Yes, Beauveria bassiana TST05 strain was identified in our previous study, so we deleted the molecular identification process of Beauveria bassiana TST05 strain and supplementary figures S1 and S2 following your suggestion.

4. The statistical difference of fungal colony numbers between layers should been marked in Fig. 1 C and the statistical analyses between the CK group and the treatment group should been labelled in Fig. 5.

Response: OK, we have finished the statistical analyses and marked them in Figure 1C. We also analyzed the significance between different layers.

The statistical analysis between the CK group and the treatment group has been labelled in Fig. 5.

5. The discussion section need to be streamlined and logic, e.g. the content same as the result section be deleted and reasonable discussion.

Response: We have simplified and modified the Discussion section, and deleted the same content as in the Results section to make the contents flow smoothly in L285~287, L288~290, L311, L331, L339, L341.

12 Jul 2022

29 Aug 2022

Dear Editor,

Thank you and the reviewers very much for reviewing our manuscript and giving us valuable comments and suggestions.

We have revised the manuscript and responded point by point to the comments as listed below. We have highlighted the changes in the revised manuscript by tracking in Word. We have also done some edits for the mistakes.

We resubmit here the revised manuscript and hope that this new revision will now be suitable for publication in PLOS ONE.

If you have any questions regarding the manuscript, please feel free to contact me.

With kindest regards,

Yours Sincerely,

Yingping Xie

Responses to the comments:

1.Why the earthworms were maintained in sterilized soil with 40 cm soil thickness? Eisenia fetida is epigeic (0-20cm) and rarely you will find it burrowing in deep soil.

Response: In this experiment, we used a 40cm thick soil layer to raise earthworms, which aimed to make the soil have better water retention capacity, so that earthworms could survive under stable humidity and temperature.

2.Line 40-42, why emphasize entomopathogenic nematodes?

Response: Thank you for your question. We have revised this part as “It was reported that earthworms may serve as phoretic hosts to entomopathogenic nematodes and B. bassiana. However, it is difficult for entomopathogenic nematodes to survive when they pass through the gut of earthworms, and the transmission of entomopathogenic nematodes by earthworms mainly depends on soil churning and mixing [5-8]. However, there is no report on the questions: Whether B. bassiana can survive when passing through the gut of earthworms, and whether earthworms can spread B. bassiana by excreting earthworm casts?” in line 36-42.

3.Line 50, how could the author draw a conclusion that a nonhost organism of B. bassiana, is there any evidence?

Response: Thanks for your question, we have revised this sentence as “However, little attention has been given to the impact of earthworms, one of the largest biomass animals in the soil, on B. bassiana [1]”in line 50-52.

4.Line 55-57, the sentence is confused.

Response: We have changed the description of the sentence as follows: “The insect A. hetaohei is an important pest of walnut fruit in northern China. The mature larvae of A. hetaohei exist in the same soil environment as earthworms and B. bassiana for 8-9 months after entering the soil.” to “The insect A. hetaohei is an important pest of walnut fruit in northern China. The mature larvae of A. hetaohei enter the soils and coexist with earthworms and B. bassiana for 8-9 months.” in line 57-58.

5.Line 57-62, the life history of insect A. hetaohei should be deleted.

Response: We have deleted Line 58 - 63 on the lifecycle of A. hetaohei. Thanks.

6.Line 64-68, more like within the scope of method.

Response: Thanks for your suggestions. We have revised this sentence as “It was reported that a high infection mortality of mature larvae was achieved using B. bassiana strain TST05 to infect A. hetaohei which was applied in walnut orchard soil [18].” in Line 61-63.

7.Line 69-77, more like the results of previous study.

Response: Thank you for your advice. We have revised these sentences as “Beauveria bassiana TST05 strain is a highly pathogenic strain that was originally isolated in 2009 by our laboratory from the naturally infected overwintering larvae of Carposina sasakii (Matsumura) (Lepidoptera: Carposinidae) in the soil of apple orchards in Xiangfen County, Shanxi Province, China. The strain was identified as B. bassiana by molecular technology [19].” in Line 65-68.

8.In this section, please clarify: what gap are you trying to tackle? And why it is important to study the effect of earthworm on distribution and pathogenicity of Beauveria bassiana?

Response: Thank you for your suggestions, we have changed the sentences to “The aim of the study is to learn how to influence the diffusion of B. bassiana in the soil by the earthworm action behavior, how to influence quantity and vitality of B. bassiana by earthworm feeding and digestion, and whether the viable B. bassiana spores in the casts of the earthworms still keep infectivity to the target insects. More importantly, we aimed to understand whether the activities of earthworms in the soil will have an impact on the number, distribution and pathogenicity of B. bassiana, and further provide a basic reference for the impact of earthworms on biological control and the application of B. bassiana as a biological pesticide.” in Line 77-83.

9.The Methods section are not well justified. Line 125, 131, etc., what is the status of sampled soil (dry or wet)?

Response: Thank you for your question, We have added the description of the soil in the manuscript in Line121-123, “The soil sample was obtained from each section tube and placed into Petri dishes. The soil block was crushed and kept for 15min. Then, 1g dry soil was weighed and diluted 10,000-fold with sterile water.”

Line128-129, “A total of 4 kg dry sterilized soil was inoculated with 640 mL spore suspension (2.5×106 spores/mL) of B. bassiana strain.”

10.Line 119-120, how about the light condition during culture period?

Response: We have added the description of the light condition in the manuscript in Line116-117, “Then, the soil columns were maintained at 25oC with 75% RH and a photoperiod of 16:8 h (L: D) for 7 days.”

11.The effective counting concentration is the dilution concentration corresponding to the plate colony count of 20 to 200. Line 126, all samples use the same dilution ratio of 10,000, the CFU of B. bassiana in CK is scientifically inaccurate (Fig.1C).

Response: Thank you for your suggestions. If different dilution concentrations are used for different experimental samples, there is a concern that the experimental error will also be increased. Therefore, the samples were diluted in the same ratio to reduce the error by repetition. Moreover, the purpose of this test is to compare the changes of spore distribution in the presence of earthworms, but not to accurately understand the specific number of spores. Therefore, the same dilution ratio was used in the test of the diffusion effect of earthworms on B. bassiana spores in soil.

12.In addition, what is the diameter of the Petri dish? The addition of one milliliter mixture for each plate is excessive.

Response: Thank you for your comment. The diameter of the Petri dish we used was 15cm. Adding too much mixture may make it difficult to coat the mixture evenly, but after the comparison of pre-experiments, it was found that adding 1ml of mixture did not have an impact on the experimental results. In order to facilitate observation, statistics and calculation, 1ml of mixture was added in this test, and the experimental effect was feasible.

13.The soil column used in Fig.1a is not described in Method.

Response: Thank you for your advice. We have added the description of the soil column in Line108-112. “PVC pipes with a diameter of 17cm and a height of 20cm were divided into four equal parts with each part 5cm high, and the four equal parts of PVC pipes were glued together with adhesive tape. The motion range of the earthworm E. fetida was confined to the soil column that was stacked with four-section polystyrene plastic tubes. ”

14.Line 219-220, is it due to the dilution ratio of 10,000 is too large?

Response: Thank you for your suggestion. This was pre-tested. When diluted 5000 times, the number of colonies in the first layer of the control group was too large to be counted, and no Beauveria bassiana colonies were observed in the third and fourth layers of the control group. If the dilution ratio of each layer is different, the experimental error might be increased, and the change of colony number cannot be seen intuitively from the picture. Therefore, 10000 times dilution was selected in this experiment. In order to reduce the error caused by dilution, this test was repeated 3 times. In each test, the experiment of the colony numbers was repeated 5 times. According to the test of Shapiro Ilan and Brown, the statistical chart showed that with the presence of Beauveria bassiana but with the absence of earthworms in the soil column for 7 days, the corrected mortality of insects in the soil of third and fourth layers was 0, which was consistent with the conclusion of this experiment. The purpose of this test is to show that with the presence of earthworms, more Beauveria bassiana spores appear in the deeper soil. Even after 10000 times dilution, Beauveria bassiana colonies can be observed in the Petri dishes, while after diluting the soil of the control group in third or forth layer, Beauveria bassiana colonies can not be observed. It was speculated that the number must be less than the group with the presence of the earthworms.

15.Line 155-157, does the midgut material contain intestinal tissue? What components contain in the supernatant? Results in 3.2 showed that great damage occurred in the midgut of the earthworm, and the colonies was conducted based on the midgut contents of the earthworms (without excreting casting). Why collect midgut material after excreted casting? Line 159, Why incubated temperature set as 4°C, far below the ambient temperature for earthworm growth?

Response: Thank you for your suggestions. In this test, the collection of midgut fluid is based on the experiment of Byzov et al.[3]. In the midgut fluid experiment, the supernatant after centrifugation of midgut tissue was used, which may contain a small amount of midgut tissue. At present, there are few reports on the specific components of earthworm midgut fluid, which needs further research and exploration. After earthworm casts, the midguts are cleaner and easier to collect midgut fluid. Intestinal juice is placed at 4ºC in order to keep the midgut fluid fresh. However, due to the reviewer's doubts about Penicillium on the plate, for the precision of this manuscript, all words and pictures related to the midgut fluid experiment have been deleted.

16. I found quite difficult to discuss inhibitory effect of the midgut fluid on the spore germination in Fig.3 since the plate is seriously contaminated.

Response: Thank you for your suggestion. The Penicillium contamination in this test was not caused by the error of experimental operation, but because, in order to collect enough midgut fluid of the earthworms, hundreds of the earthworms were dissected and the midguts were collected in this experiment, and, it is inevitable that there were other miscellaneous microbes in earthworm midguts. Even before using earthworms as experimental materials, raising them in a sterile environment and make them cast cannot remove all miscellaneous microbes in the midguts. Therefore, after mixing the midgut fluid with Beauveria bassiana suspension, Penicillium in the midgut fluid will also grow on the Petri dish and form colonies. After many repetitions of this test, Penicillium colonies were inevitably found in the Petri dish. According to the suggestion of the reviewer, For the precision of this manuscript, all words and pictures related to the midgut fluid experiment have been deleted.

17. The MS requires language editing. Some sentences are awkward. (Line 92; Line 103-104, and many more…)

Response: We have checked all the main text and did some edits.

BASIC ERRORS:

1.Corresponding Author name (see Manuscript Draft in submitted PDF)

Response: Have Corrected the Corresponding Author name

2.Latin name (A. hetaohei Yang and A. hetaohei; E. fetida and E. foetida; full name and abbreviation of the Latin name)

Response: Have Corrected Latin name

3.The use of Arabic numerals. They should never be used at the beginning of sentences. (Line 167)

Response: Have corrected in line 151.

4.Keywords should not be words found in the title. More, exceed the required number of keywords.

Response: The keyword is modified to "Eisenia fetida, cast, TST05, pathogenicity, spore germination, Infection, Insect, Entomopathogenic fungi".

5.Segmentation is confusing (Line 179-191)

Response: Have corrected in Line 164-175.

Submitted filename: Response letter8.20.wps

Click here for additional data file.

26 Sep 2022

Distribution and pathogenicity of  Beauveria bassiana  in soil with earthworm action and feeding

PONE-D-21-30327R2

Dear Dr. Xie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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3 Oct 2022

PONE-D-21-30327R2

Distribution and pathogenicity of Beauveria bassiana in soil with earthworm action and feeding

Dear Dr. Xie:

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