| Root-knot nematodes are very damaging soilborne pathogens that hurt root growth and development and subsequently block utilization of nutrients and water. The symptoms of root infection by root-knot nematodes can be always observed as color of leaves changed from green to yellow and huge numbers of root-knots and stunted plant growth as well as decreased yields. Economic and crop loss has been reported all over the world in every year. Continuous cropping system is a main reason for incidence of root-knot nematode. However, once a crop has been infected, the control by crop rotation is very difficult because a wide of plants including most common crops and weeds can serve as hosts of root-knot nematode. Several chemical nematicides can effectively control root-knot nematode, but their use is strictly limited and even banned in the world for they cause serious pollution of environments and greatly damage to human health. Therefore, exploration of new and environment-friendly bio-control alternatives is very urgently needed. The main contents and results in this paper are listed as follows:(1) Experiments on enrichment, isolation, purification and identification of pathogenic root-knot nematode. Root-knot nematodes were enriched in greenhouse by growing a susceptible tomato variety in soil from heavily field by root-knot nematode infected. Root-knot nematodes species were isolated from roots by combination of the Baermann funnel, sieving and tissuse-mashed methods. The healthy soil was re-inoculated with the isolation purify the root-knot nematodes, which was then identified by morphological characteristics under microcopy. Both the enrichment and re-inoculation experiments showed that the number of root-knot nematode in soil was increased by range from1.6to3.3times as compared with the infected field soil and the root-knots increased with a4scale of galling index grade while roots of tomato grown in healthy soil without root-knot nematodeor inoculation of root-knot nematode were not infected. The females, second-stage juveniles and eggs of nematodes were isolated from infected roots. The nematode was eventually identified as Meloidogyne incognita species according to its morphological characteristics. Thus, the all results indicated that the obtained root-knot nematodes could grievously caused damage to tomato plants and be used for the next experiments in future. The eggs embryo development of root-knot nematode were then observed under microscope for14days at25℃. The results showed that eggs embryo development stages included single-cell stage, two-cell stage, three-cell stage, four-cell stage, blastula, gastrula stage, first stage juvenile, first molt of second-stage of juvenile, second molt of second-stage of juvenile, and second-stage of juvenile after molt.(2) Screening bio-control agents. Collagen is main component of root-knot nematode cuticle, so based on this theis the rhizosphere bacteria that had ability to produce collagenase are isolated from soil. Our protocol included:firstly to isolate collagenase-producing bacteria from rhizosphere soil of tomato by dilution-plate method, to test the effect of culture filtrate of strains on nematocidal activity and eggs hatching under in vitro experiment, and then to investigate effect of bacterial suspensions on root-knot nematodes infection of tomato in greenhouse. By this procedure, we could screened high effective biocontrol rhizosphere bacterial strains and understood the action mold of the interaction between the bacterial strains and root-knot nematodes. Ten bacterial strains with strong ability to hydrolyze collagen were isolated. Among these strains, strain X5and strain BTG were best with high collagenase activities of40.5U·mL-1and37.3U·mL-1, respectively. Compared with the control treatment of only water, supernatants of strain X5and strain BTG increased J2mortality rates by73.1%and60.2%, respectively, after24h of incubation, while decreased hatching rates of nematode eggs by37.7%and25.6%, respectively after14days of incubation. As the concentration of supernatants of both strain X5and strain BTG was decreased in the order from1,1/2, to1/4of the initial fermentation liquid, the mortality of J2was decreased while hatching rates of eggs were increase In greenhouse, inoculation of root-knot nematodes diseased soil with cell suspensions of strain X5and strain BTG reduced the number of tomato root-knots in soil by79.5%and64.8%, respectively, as compared with the non-inoculation control. Analysis of16S rDNA sequences showed strain X5and and strain BTG belong to Bacillus cereus and Bacillus thuringiensis, respectively. Comparison of the effect of the two strains on the J2mortality, eggs hatching rates and root-knots numbers showed that strain X5was more effective in inhibition root-knot nematodes than strain BGT, indicating that X5had potential as a bio-control agent. Microscopically observation showed that the inner structural protoplast of most eggs were destroyed, disordered and vacuolatedafter addition of the supernatant of strain X5. The vacuolation rate decreased with the embryo development from eggs to J2. Thus, the strain X5had ability of not only killing the J2but also destroying the eggs of root-knot nematodes.(3) The character of collagenase and its nematocidal activity. The collagenase was purified from fermentation production of strain X5through three steps procedure involving ammonium sulfate precipitation, gel-filtration-Sephadex G100column and anion-exchange chromatography. The purified enzyme exhibited the strongly collagen hydrolyzing activity on plates. The initial specific activity of the protease was13.0U·mg-1, but the final of that was153.4U·mg-1. This suggested that the purification efficiency was increased by11.8times. The recovery rate was9.3%of fermentation liquid. The molecular mass of the protease was45kDa, as determined by SDS-PAGE. As comparison with controls, the mortality of J2of root-knot nematodes reached to80%and the egg hatching rate decreased to23.9%after treated with purified collagenase enzyme. Analysis with scanning electron microscopy showed that both the cuticle of J2and the eggs surface were damaged and the protoplasm of eggs was disordered after addition of the enzyme. The characterization of the purified collagenase indicated that optimal pH, optimum temperature, pH stability, and thermal stability for its activity were pH8.0,35℃, pH7.0-10.0, and10℃-40℃, respectively. Existence of metal ions Mg2+、Zn2+、Fe2+and Ca2+increased positively enzyme activity, while addition of Cu2+、Ba2+、Fe+and Mn2+inhibited the enzyme activity. It was found that Ca2+ion could strongly activate enzyme activity. The enzyme became almost inactive after addition of phenylmethanesulfonyl fluoride (PMSF), implied that the protease is a serine protease. However, ethylenediaminetetraacetic acid (EDTA) had little inhibition on the collagenase activity. It is concluded that this enzyme from strain X5had nematocidal activity to kill nematodes through degradation of their cuticle and reduction of egg hatchability.(4) The ability of strain X5to colonize in soil and roots. The plasmid Phapii GFP with Pseudomonas promoter and GFP segment was transformed into strain X5by electroporation and we gained labeled strain X5-gfp. X5-gfp colonized on tomato root and in soil were obversed by using fluorescent microscope and freeze-slice up methods, while mortality of J2and egg hatching rate of RKN were tested in vitro. After X5-gfp grew for20to25generations, the stability of plasmid was in the range from76%to60.5%, suggesting that the plasmid could retain rather stably in strain X5-gfp. The growth curve and physiology characteristic of labeled strain X5-gfp were not changed, while the mortality of J2increased by66.3%and the egg hatching rate was reduced by65.1%after treated with culture filtrates of strain X5-gfp. Observation by fluorescence confocal microscopy showed that the population of strain X5-gfp in bulk soil, rhizosphere and rhizoplane was increased in order as follows:rhizosphere> rhizoplane> bulk soil. The population of strain X5-gfp in bulk soil was6.6×108cfu·g-1after inoculated7days. The galling number of tomato reduced by63.1%after35days of growth as compared with the control. Thus, strain X5could protect plant from infection by RKN through effective colonization of tomato roots and soil.(5) Effect of bio-organic fertilizers fortified with strain X5and strain BTG or both. Or both with a bio-organic product (BIO) on control root-knot nematodes of tomato in greenhouse pot experiments. The height of tomato plant, egg masses per plant and egges per egg mass were assayed, and the total number of nematodes and micro-organism (bacteria, fungi and actinomycetes) in the rhizospherer soil of tomato plant were counted50days after application of bio-organic fertilizers. As compared with the control, application of the X5or the BTG bio-organic fertilizer or the compound (X5+BTG) bio-organic fertilizer increased the dry weight of shoot by0.5-0.9times, and dry weight of root by0.7-0.9times. Egg masses per plant and eggs per egg mass also reduced by24.4-54.2%and21.8-54.3%, respectively. The total number nematodes in rhizosphere soil of tomato reduced by18.6-49.2%. However, application of the compound bio-organic fertilizer (BIO+X5+BTG) gained best results as the dry weight of shoot and root of tomato plant increased by2.1and1.5times, respectively, and egg masses per plant, egges per egg masses and nematodes of rhizosphere soil reduced by67.2%,62.1%and50.7%, respectively, as compared with the control. The field experiment indicated that application of the bio-organic fertilizer enhanced by strain X5(treatment X5) or the mixed bio-organic fertilizer (treatment BIO+X5+BTG) could effectively control the root-knot nematodesof muskmelon through reduction of the galling index of muskmelon by77.6%and81.6%, respectively, as compared with the control. These result indicated that the functional bio-organic fertilizers could control root-knot nematodes disease and promote tomato growth. Thus, strain X5might play an important role in the related bio-organic fertilizers.(6) Integrated root-knot nematodes control management. Greenhouse experiments were conducted to evaluate the effect of film-covered solarization of soil, biofumigation with organic materials and inoculation with antagonistic organisms on control root-knot nematodes of tomato. Plant biomass, root-knot nematodes in soil and plant, and microbial community in bulk and rhizosphere soil were determined. Results showed that combination of film-covered solarization of soil, biofumigation with organic materials and inoculation with antagonists gained the best biological control efficiency than all other treatments. When compared with no treated control, the combined treatment increased total biomass of tomato by1.8times, decreased egg masses of root-knot nematodes per plant and eggs in root by2times and3.7times, respectively, and gained82.7%of biological control efficiency on root-knot nematodes. The combined treatment also significantly increased numbers of bacteria and fungi, and significantly decreased numbers of nematode in both in rhizosphere and bulk soil as compared with those of the control. Therefore, application of strain X5with biofumgation and solarization could realize the synergistic effect of these bio-control measurements in efficiectly control of root-knot nematodes and regulate soil microbiological composition to safeguard plant growth in goof health. |
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