Interaction Between Earthworm And Beauveria Bassiana In Soil And Its Effect On Biological Control

Beauveria bassiana is one of the most widely studied and used insect pathogenic fungi.During the biological control of pests,B.bassiana will persist in the soil,and inevitably contact with earthworms,especially the epigeic species after application.So,how the activity,feeding and epidermal mucus of earthworms will affect the activity of B.bassiana?In this study,the effects of earthworms on B.bassiana,including the distribution,activity and pathogenicity of B.bassiana in soil,were evaluated by using the the epigeic earthworm species,Eisenia fetida,B.bassiana TST05 and mature larvae of the Atrijuglans hetaohei.The distribution,biomass and morphology of B.bassiana TST05 in soil,the spore morphology under scanning electron microscope were observed,and its spore germination rate,fungal extracellular enzyme activity and pathogenicity to A.hetaohei were detected.The results showed that earthworm activity and feeding could promote the downward diffusion of B.bassiana,but reduce the number of live spores.The midgut fluid of earthworm obviously inhibited the spore germination of B.bassiana.B.bassiana was still pathogenic to the insects after excreted by earthworms.The spores of B.bassiana could attach to the earthworm epidermis,but they would be covered by the epidermal mucus over time,and becoming rough and atrophic.The spore germination and extracellular enzyme activity of B.bassiana were significantly inhibited after treated by epidermal mucus.After inoculating the mixture of B.bassiana and mucus in the mature larvae of A.hetaohei,it was found that mucus could reduce the pathogenicity of B.bassiana to insects,thus slowing down the course of disease and reducing mortality.The results showed that earthworm activity and feeding promoted the distribution of B.bassiana in soil,however decreased the number of living spores of B.bassiana.The epidermal mucus of earthworm can inhibit the activity of B.bassiana and its infectivity and pathogenicity to target insects.However,after being treated with epidermal mucus,the surviving B.bassiana still has certain infectivity to insects.This study is of great significance for understanding the interaction between earthworms and B.bassiana in soil and guiding the scientific application of B.bassiana in biological control of pests.1.The ultrastructure of earthworm epidermis and gut treated by B.bassiana was observed.The experimental results showed that the ultrastructure of earthworm epidermis was obviously changed after the treatment of B.bassiana.The cuticle of the epidermis was thickened,the diameter of the secretory cells on the epidermis increased,the secretory vesicles expanded,and the secretory granules increased.In this experiment,the number of mitochondria in cells of earthworm foregut increased,and vacuoles of mitochondria were observed.The cilia of the earthworm midgut were thickened,and the number of mitochondria and ribosomes in midgut cells increased.There was no significant difference between the control group and the earthworm treated with B.bassiana.2.In this experiment,a four layer soil column was used to study the effect of E.fetida activity on the distribution of B.bassiana in soil.The results showed that in the control group with the absent of the earthworms,the spore of B.bassiana inoculated on the soil surface did not diffuse downward significantly.The spores of B.bassiana mainly remained in the first layer of the soil column,and the spores in the second layer were few,and did not appeared in the third and fourth layers.In contrast,the distribution of B.bassiana spores in soil column changed significantly in the treatment group with the presence of the earthworms.B.bassiana colonies were isolated and cultured from one to four layers of the soil samples.This showed that the earthworm activity made the B.bassiana spores inoculated on the surface of the soil column diffused downward to 15～20cm deep(the fourth layer).Among them,many B.bassiana colonies were isolated and cultured in the second and third layers of soil samples,indicating that a large number of spores can be spread and distributed to 10～15cm deep.In addition,the test results also showed that the total number of B.bassiana colonies isolated from the soil of the treatment group with earthworm activity was significantly reduced by about 80% compared with the control group.The results showed that earthworm activity promoted the downward diffusion of B.bassiana in the soil on the one hand,but reduced the number of active B.bassiana spores on the other hand.3.In this study,the contents of the foregut and midgut of E.fetida and the colony number of B.bassiana were determined.The results showed that some B.bassiana could survive after passing through digestive tract of the earthworm,and could be carried and spread with activities of the earthworm and the cast in the soil.However,it was also found that the total number of living B.bassiana spores would be reduced in this process.Significant changes occurred in the midgut and earthworm cast.The number of B.bassiana spores detected from the midgut content was nearly half that of the soil sample,and the number of spores detected in the earthworm cast was only 14.2% of the soil sample.This may be due to the digestion of B.bassiana spores by earthworms after passing through the gut of earthworms.Therefore,the midgut fluid of E.fetida was used to test the inhibition of spore germination and colony growth of B.bassiana.The results showed that the inhibition was very obvious,which decreased by 91% compared with the control group.The difference was very significant,indicating that earthworm gut fluid had a strong inhibition on B.bassiana spore germination.4.In order to verify the effect of intestinal digestion of earthworms on the pathogenicity of B.bassiana,the larvae of A.hetaohei were infected with the B.bassiana spores in E.fetida cast and the B.bassiana spores in soil(CK group)respectively.The results showed that the infection process and symptoms of the two groups of larvae were similar,and there was no significant difference in mortality and median lethal time.It can be inferred that,although the number of B.bassiana spores will be significantly reduced after digestion by earthworms,the B.bassiana spores surviving in earthworm cast still have strong infectivity to target insects.5.In this study,the method of impregnation was used to infect the earthworm.SEM observation showed that the spores of B.bassiana could attach to the epidermis of the earthworm,but spore germination and invasion of earthworm epidermis were not observed.The B.bassiana spore could attach to the hollow of the earthworm epidermis,and also attach to the internode groove and around the stoma.The surface of B.bassiana spores initially attached to the earthworm epidermis is smooth,round and full.As time goes on,the spores of B.bassiana will be coated by the epidermal mucus secreted by earthworms,the surface of the spores became rough and uneven,and finally became shrink and shrivel,unable to germinate.This shows that although the spores of B.bassiana can attach to the the earthworm epidermis,the epidermal mucus of the earthworm could wrap the spores,preventing the germination and subsequent invasion of the spores.Thus it can be seen that the epidermal mucus is the first barrier for earthworms to prevent the invasion of pathogenic fungi.6.In this study,earthworm epidermal mucus was extracted and prepared into a solution of 5 concentrations to treat B.bassiana spores to observe its effect on B.bassiana spore germination and colony growth.The results showed that earthworm mucus treatment did significantly inhibit the germination and colony growth of B.bassiana,and the greater the concentration,the stronger the inhibition.7.During the process of B.bassiana invading insect cuticle,the extracellular enzymes secreted by fungi also played an important role in the degradation of insect cuticle.The activity of extracellular enzymes of B.bassiana is usually considered as an important indicator of its infectivity to host insects.In this study,the treatment group EW added earthworm epidermal mucus lyophilized powder to the liquid medium every day,while the treatment group EO added earthworm epidermal mucus lyophilized powder only once at the beginning of the culture.It was found that during the germination of B.bassiana,earthworm epidermal mucus had significant inhibition on four fungal extracellular enzymes(subtilisin,lipase,chitinase and NAG),and the inhibition could last for a period of time.The results showed that earthworm epidermal mucus could inhibit the secretion of extracellular enzymes.However,if B.bassiana no longer contacts the newly secreted earthworm epidermal mucus,the inhibition will gradually weaken.8.In order to verify whether the earthworm mucus has an impact on the pathogenicity of B.bassiana,the spores of B.bassiana treated with earthworm epidermal mucus were inoculated on A.hetaohei larvae.The results showed that earthworm epidermal mucus could reduce the pathogenicity of B.bassiana to insects,resulting in slower disease course and lower mortality of larvae during infection.However,although the pathogenicity of B.bassiana to A.hetaohei larvae is affected by the epidermal mucus of earthworms,the B.bassiana spores which surviving after treatment with the earthworm epidermal mucus still maintain a certain infectivity to insects.In addition,with the loss of active components in earthworm epidermal mucus,the inhibitory effect of earthworm epidermal mucus on B.bassiana will gradually weaken after 5-7 days.9.In order to understand whether B.bassiana has an impact on the natural immunity of earthworm epidermis,DEGs related to the secretion of earthworm epidermal mucus and antibacterial effect were screened from transcriptome data.It was found that a variety of genes related to natural immunity were differentially expressed,indicating that overexpression of these genes in earthworms may be a positive reaction to the attachment of B.bassiana,which is related to its complex immune mechanism.