Toxicity On Nematodes And Mechanism Of 5-aminolevulinic Acid Produced By Photosynthetic Bacteria

Plant-parasitic nematode is one of the important pathogen in agriculture and cause serious crop losses worldwide, and become an important constraint to modern agriculture. The current method to control nematodes.is largely depend on chemical nematicide. However, the chemical nematicide is highly toxic to human and the control effect is more and more decreasing because of pathogen resistance Recently, many scientist give more concerns to bioagents on controlling of nematodes because of its low toxicity to human and low resistance risk to target.In this study, the nematicidal activity and control effect of Rhodoblastus acidophilus HB-1 to nematode were assayed. Meanwhile, the nematicidal activity of 5-aminolevulinic acid (ALA), a metabolite of HB-1, were tested. The nematicidal mechanism of ALA was deduced through nemetode morphology, biochemical test, EM results. The gene of ALA synthese was cloned from HB-1 and further expressed in E. coli for ALA production. Our work make the basis to develop biological control agents against nematodes. The detailed results are as follows:1. In this work, bioassay of the culture fluid of HB-1 against C.elegans and 2nd stage juveniles of M. incognita were carried out. The results showed that HB-1 has high nematicidal activity with a lethal concentration (LC50) value of 5.13?L/mL and 4.81?L/mL after 96h incubation respectively. Subsequently, the control efficiency of HB-1 against root-knot nematode in greenhouse and in field were checked. The results indicated that HB-1 could efficiently inhibit the infection of M. incognita to tomato and cucumber. At the same time It could also obviously reduce the formation of galls and significantly promote the plant growth. The control effect of HB-1 to M. incognita is 71.5% and the yield of cucumber increased 57.7%. The results showed the great potential of HB-1 on controlling of the plant-parasitic nematodes.2. The nematicidal activity of ALA against C. elegans, M. incognita, H. glycines and B. xylophilus were tested. The results showed that ALA is high nematicidal activity to many kind of nematodes and has a broad spectrum activity. The LC5o were 162.5mg/L,188.5mg/L and 442.2mg/L of M. incognita, H.glycines and B. xylophilus after 96h of incubation, respectively. The eggs of M. incognita were inhibited to hatch and the rate of hatching reduced 70% at concentration 500mg/L compared with control. Based on those results, we tested the control efficiency of ALA against root-knot nematode in greenhouse.The results showed that ALA could efficiently inhibited infection of M. incognita to tomato in greenhouse, the formation of galls were obviously reduced and the number of nematodes in root was decreased. The control efficiency was 81.7% at concentration 40mg per plant and was comparatively with Avermectin.The nematicidal activity of ALA under light condition is increased, howerev, the pH and temperature is no effect to its activity.3. The mechanism of ALA to nematodes was studied. We observed the morphological changes by microscopy and detected metabolites of nematodes. The nematodes, which were treated by ALA, became active, then stiff, the organization dehydrated, cell structure destructed and similar to autophagosome. ALA could interfere the metabolism of protein, induced SOD,CAT,GSH-Px activity and increased MDA. The SOD,CAT, GSH-Px activity and MDA of treatment is 1.78,2.33,1.35 and 2.70 times to the control groups respectively. ALA could induce the AchE activity at low concentration while inhibit at high concentration. Taken together, our results indicated that ALA could induce the free radical to destroy the protective enzyme system, subsequently lead to lipid peroxidation, cell structural damage, membrane permeability change and finally contribute to nematode death.4. The ALAs gene of Rhodoblastus acidophilus was cloned from its genomic DNA by Veterette PCR and further sub-cloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAs was expressed in Escherichia coli strain M15. The expression condition including strain, medium, substrate of ALA synthesize (glycine and succinic acid) and ALA dehydratase inhibitor (levulinic acid) were optimized to achieve the maximum yield of ALA in E. coli M15, and the maximum output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. The nematicidal products were manufactured through formulation process. And the control efficiency of this products against root-knot nematode in greenhouse was tested, the results showed that the products could efficiently inhibit the infection of M. incognita to cucumber, obviously reduce the formation of galls. The control effect to root-knot nematode was 76.67%, which is better than Avermectin-triazophos. Our results indicated that this product has great potential on controlling of nematodes.