The Inhibiting Effect Of Beauveria Bassiana Induced Solanum Lycopersicum Defense On Bemisia Tabaci

Induced defense is an important part for plant defense strategies.Plants recognize multiple signal molecules to initiate their own defenses to deal with pest infestation.With the deepening of research,it was discovered that Beauveria bassiana can serve as a plant endophyte and share the same host plant with insects as a habitat.Endophytic fungi are often closely related to plant defense systems and play an important role in controlling pest populations.However,endophytes have been ignored by people for a long time.Revealing the interaction between B.bassiana and host plants,improving plant resistance to insects,can provide new strategies for biological control of pests.This article takes B.bassiana,Solanum lycopersicum and Bemisia tabaci?biotype:Q?as research objects,aims to clarify the mechanism of action between B.bassiana–tomato-B.tabaci,exploiting the application potential of B.bassiana as a plant endophytic fungus to induce tomato resistance and its related control mechanisms.Through relevant research,the research results were obtained as follows:1.Study on colonization rules of B.bassiana in tomatoThe sequences of B.bassiana in S.lycopersicum plants were obtained by Nest PCR,which confirmed that B.bassiana could enter the plants by artificial inoculation.By comparing the three methods of artificial inoculation,it was found that foliar spraying had the highest colonization efficiency.At 7 days after inoculation,colonization rate increased significantly,and in 14 days later,the colonization rate reached 100%,followed by root-irrigation inoculation and seed-dressed in lowest.Selective culture medium was used to isolate B.bassiana in the plant tissue,and it was found that the fungi were distributed systematically in the plant,but not evenly,preferring existing at the inoculation site.When the spraying concentration was 1×10⁸ conidia/mL,the number of colonies isolated from the mesophyll tissues reached 8.5±2.02,while root-irrigation was adopted,a large number of colonies could be separated from the roots and stems of plants.The determination results of the main growth index of plants showed that the colonization of B.bassiana had no negative effect on plant growth and even promoted plant growth at certain concentrations.The principal component analysis showed that leaf spraying inoculation had a stronger promoting effect on plant growth.When the concentration of spores was 1×10⁵ conidia/mL,the effect of spraying inoculation had a more significant effect on plant growth,with the highest contribution,that was 2.645,which had the most significant promoting effect on root growth index?characteristic value=2.228?but had a minimum effect on the weight of dry matter?characteristic value=0.404?.2.Study on the tomato defense path of B.bassiana-inducedQuantitative detection of tomato defense genes after inoculation with B.bassiana,the results showed that inoculation of fungi could increase the expression level of plant defense genes.The defense genes of S.lycopersicum plants significantly increased after7 days,which reached to the most.Especially after 7 days,phenylalanine ammonia lyase?PAL?gene up-regulated at 18.52 times that reached the peak.Co-expression analysis of defense genes at different time nodes showed that four genes?PIN2,PR2,PAL,MPK3?were up-regulated at all time points and participate in the transduction and transmission of plant defense signals.The expression profiles of genes related to defense of S.lycopersicum were significantly different under the combination of fungi induction and insect feeding.There were totally 9 genes up-regulated,which mainly involved defense-related genes in the primary and secondary metabolic pathways of plants,among which PAL,PPO,PIN2,PR2 and PR1 genes were all related to the synthesis of phenolic compounds.In addition,the identification of plant metabolites by GC-MS showed that plants always gave priority to the synthesis and metabolism of their own nutrients.For example,the needs of energy for their own growth and development were met through synthesis of gluconeogenesis,fatty acid,amino acid and other ways.After the stimulation induced by B.bassiana,the content and species of plant secondary metabolites increased significantly.Under the mode of fungal induction,19 defense-related pathways were obtained by KEGG database annotation,and the defensive products were mostly enriched in the phenylpropane metabolic pathway mediated by PAL.Under the regulation of the defense pathway,plants could produce various allelopathic substances,among which phenolic substances were the main defense end-products.In addition,the content differences in the jasmonate,a hormone that regulated plant defense,and its synthetic precusor substance,?-linolenic acid and I-isoleucine were detected,and the contents were significantly increased after fungal induction.This also shows that under this induction condition,plant defense is mainly regulated by the jasmonate hormone network.3.Effects of induced defense reaction on the developmental period of B.tabaciS.lycopersicum was pre-inoculated by spraying and root-irrigation respectively,and the plants inoculated with B.bassiana would have a great influence on B.tabaci population.Based on comparison among the mortality of each instar,it was found that the mortality of eggs and young nymphs was higher.As for the plants inoculated with spraying,the mortality rate of B.tabaci eggs reached more than 50%.However,the eggs mortality rate was only 42%when inoculated by root-irrigation.In addition to the effect on the population mortality of B.tabaci,the feeding fungi pro-inoculated plants could significantly prolong the development period of individual B.tabaci.Compared with16.39 d after fed on healthy plants,the developmental period of the B.tabaci was extended to more than 20 days on average after fed on fungi spraying-inoculated plants.Especially at the inoculation concentration of 1×10⁸ conidia/mL,the developmental period of whitefly was extended to 21.45 d.As for the specific age,the egg stage and the first instar nymph stage were significantly prolonged.After root-irrigation to the foot end,the developmental period of B.tabaci after fed on the upper leaves was also significantly prolonged,reaching 20.34 d.It was further confirmed by PCR detection that the B.tabaci fed on the inoculated plants were not amplified any B.bassiana sequences.In addition,tomato mutant material was used to verify the plant defense effect.After detection,the mutant material inoculated with B.bassiana did not activate the plant defense level.Most of all,amount of gene expression of PAL and LOX related to jasmonic acid regulation were lower than those of control plants.While the difference between the mortality rate and the developmental period of whitefly after fed on the mutant material was not significant,and the developmental period of B.tabaci population was only 18.65d,which indicated that the survival rate and development of B.tabaci population were mainly affected by plant defense.4.Inhibition effect of induced defense reaction on whitefly digestibilityAfter determining the effect of plant defense reaction on the development of B.tabaci,the damage mechanism was further analyzed from the perspectives of physiology.The detection results of the digestion gene were verified that the plant defense reaction would inhibit the genes of sucrase,amylase and protease.In both short-term and long-term feeding,digestive gene expression showed a downregulation trend and continued to be down-regulated even after the insects fed on normal food at the later stage.In addition,the main digestive enzyme activity and the expression of digestion genes of B.tabaci also showed a decreasing trend.The activity of three main digestive enzymes significantly decreased?amylase activity decreased by 1.31 U/dl,trypsin activity decreased by 23.65U/mg prot,and sucrose activity decreased by 0.12 U/mg prot?.Total activity of digestive enzyme and total digestive matter decreased by 1.64 U/g and 168.9 ng/g,respectively.The vitro experiment of the plant extract mixed with the digestive enzyme standard showed that the plant extract had the same inhibitory effect on activity of various digestive enzyme.However,the lack of digestive ability will lead to the decrease of individual nutrition level.In addition that there was no significant difference between the glucose content and that of the control group,the fresh weight,dry weight,total sugar,total protein and amino acid content of B.tabaci showed a decreasing trend,especially the total sugar content and amino acid content decreased to 0.023 mg/g and 177.0?mol/mL,respectively.Under the stress of tomato defense function,the main digestive function of B.tabaci was inhibited,and the individual nutrition level began to decline.5.Evaluation of intestinal damage of B.tabaci feeding on inoculated plantsHistopathological observation,immunofluorescence labeling was carried out on the target sites of plant defense reaction in insects to explore the damage type of intestinal tissue and cell vitality.Histopathological observation showed that the adult insects fed on the inoculated plants had loose breast tissue and tissue surface structure became irregular.In the intestinal tissue,the cells also changed from a compact form to a loose discrete state,accompanied by significant vacuolization.Changes of Ca²⁺and reactive oxygen species also predicted activation of tissue damage signal.After fed on the inoculated plants,the fluorescence signal of Ca²⁺in the intestinal tissues of B.tabaci was particularly obvious,and the reactive oxygen species increased rapidly from 790 U/g to 1300 U/g.Stimulation due to damage signal,the detoxification metabolic genes of B.tabaci showed up-regulated in the initial feeding period.However,as time goes on,that cytochrome P450,glutathione s-transferase?GST?and carboxylesterase genes were become lower than that of the control insects.Therefore,intestinal cell activity was significantly inhibited,and the cell activity value was decreased by 0.2153.With the increasing damage,the body began to regulate apoptosis,while Caspase-3 was a kind of enzyme in the Caspase family responsible for apoptosis.In the intestinal cells of B.tabaci,the content of Caspase-3 increased rapidly,reaching 0.6864 mM.The nuclear staining also showed increased cell damage and a stronger fluorescent signal in the B.tabaci fed on the inoculated plants.Finally,there was an irreversible effect on tissue permeability,and a large number of FITC-Dextran markers could penetrate intestinal tissue,which indicated an increase of permeability of intestinal tissue.6.Evaluation of the inhibitory effect of tomato induced defense reaction on B.tabaci populationIn addition to prolonging the development of B.tabaci,tomato induced defense could also lead to the effect of eluding from feeding of B.tabaci.The randomized selective test showed that the adult preferred to feed on uninfected S.lycopersicum plants.When processing under the concentration of spores was 1×10⁸ conida/mL,1×10⁷conidia/mL and 1×10⁴ conidia/mL,the number of spawning on the leaves of the treatment group was 7.25,45.50 and 16.25,far lower than that of the control group.Selective tests using plant extracts showed more significant differences,which indicated that the B.tabaci apparently preferred uninoculated plant extracts?80.5%?.In addition,due to the stress of plant defense ability,the disease resistance of B.tabaci population to entomopathogenic fungi decreased.The lethal concentrations of B.bassiana to the two B.tabaci populations were significantly different.The LC₅₀ of the normal B.tabaci population was 3.81×10⁷ conidia/mL,while the LC₅₀ of the B.tabaci population under plant defense stress was 1.55×10⁷ conidia/mL,causing various adverse effects on B tabaci population.This mode of using pathogenic microorganisms of exogenous insects to mediate plant hosts to jointly control agricultural and forestry pests often has the advantages of rapid development,resistance to adversity,disease resistance and anti-arthropod hazard,etc.Fungus-colonized plants were more competitive for survival than uninoculated plants,damaged insect digestive system and causing an adversely effect on pest populations.As a result,the endophytic flora plays an unprecedented important ecological role in the ecosystem of endophytes-plant hosts-agricultural pests.