| Banana(Musa spp.)is one of the most important crops in the world as a staple food,and also the main source of income for local farmers in tropic areas.However,the production of bananas is hampered by many diseases and pests.Among the most damaging banana pests are the widespread plant-parasitic nematodes.In particular,the root-knot nematodes,Meloidogyne spp.,are economically important soil-borne pathogens that are reported to infect almost all of the world’s major crop plants.Nematode management is traditionally achieved by application of chemical nematicides.However,growing concerns about food and environmental safety have led to the withdrawal of several commonly used chemical nematicides.Effective control strategies for Meloidogyne javanica may include the use of plant-origin nematicidal agents.Camellia seed cake is an organic substance made of the residual from Camellia oleifera Abel seeds which were used for oil extraction.In this study,the nematicidal ability of Camellia seed cake and some of its potential mechanisms were investigated in detail to explore a green strategy for managing M.javanica in banana planting.Effects of Camellia seed cake application on the soil chemical properties,soil nematode communities and microorganism communities were studied to clarify the mechanisms of Camellia seed cake application in suppression on root-knot nematodes of banana plants.1.Isolation and identification of M.javanicaRandom visible suspect Meloidogyne spp.Females and egg masses were collected from individual banana(Musa AAA Cavendish cv.Brazil)seedlings with serious banana root knots.Two obvious lateral lines were observed in the perineal patterns of the females,and the hyaline tail of juvenile.Approximately 670 bp band was detected in most of the Meloidogyne spp.second-stage juveniles(J2s)using the SCAR primer Fjav/Rjav.After that,these Meloidogyne spp.were identified as M.javanica based on the phylogenetic tree analysis and morphological identification.Re-inoculation of these Meloidogyne spp.into the banana plants produced banana root nematode disease.The isolated M.javanica was confirmed to be the dominant species of banana root-knot nematodes in this research.2.Nematicidal effect of Camellia seed cake chemicals on M.javanicaA juvenile corrected mortality of approximately 100%was observed and approximately no juveniles were hatched after treated with 5 g/L of Camellia seed cake.After treated with 5 g/L of Camellia seed cake,the cuticle of the juvenile was crimpled and the intestine was obviously dissolved at 72 h;the eggs showed the content was apparently dissolved and the egg shell layers was obviously reduced.Meanwhile,the bacterivores and fungivores corrected mortality of approximately 100%was observed after treated with 1 g/L of Camellia seed cake.Saponin homologues of 1240.50 Da in Camellia seed cake extract were detected and identified by HPLC-ESI-MS,which showed nearly 100%corrected mortality at 50 g/L of Camellia seed cake.Volatile compounds from Camellia seed cake also showed a nematicidal effect.18 compounds in volatile compounds were identified by GC-MS.8 types of volatile compounds acquired exhibited effective nematicidal activities,especially 4-methylphenol,which showed highest efficiency and yielded a corrected mortality of 100%with an added amount of 30 mg/L.3.Nematicidal effect of Camellia seed cake on root-knot nematode in pot experimentThree banana growing seasons of pot experiments were performed for the Camellia seed cake effect on M.javanica.For the first-season,Camellia seed cake was added in the sterilized soil in each pot which was inoculated with juveniles of M.javanica.Sixty days after transplant of banana seedlings we collected soil and plant samples to analyze the effects.Compared to the control without Camellia seed cake,application of 5 g/kg of Camellia seed cake significantly increased the plant biomass,decreaed the eggmasses per plant and the eggmasses per gram root.For the second-and third-seasons,a banana field soils with severe nematodes was put into pots and Camelia seed cake was added to evaluate its nematicidal effect.Sixty days after transplant of banana seedlings,we did the analysis of soil and plant samples and got similar result with the first season.Application of 5 g/kg Camellia seed cake increased the nematode density by 60.15%.With identification of the nematodes using morphology and feeding-habit based classification,the numbers of plant parasites in Camellia seed cake treatments were significantly lower than those in the control,while the numbers of fungivores and bacterivores were significantly greater when compared to the control.For the culturable microbes,the numbers of bacteria,fungi and actinomycetes of soils in Camellia seed cake treatments were all significantly higher than those in the control.Considering the high nutritional value and essential elements contained in Camellia seed cake,microbes biomass was increased.The fungivores and bacterivores abundance could be increased by the nutrition and microbes.For the third-season,similar results for the growth parameters were observed as the second-season.The results from all the pot experiments showed that the application of Camellia seed cake not only suppressed M.javanica but also promoted the banana plant growth.4.Exploring Camellia seed cake applied patterns for soil nematode communitiesTo explore the effect of Camellia seed cake applied patterns on soil nematode assemblages and microbial abundance,soils in the treatment added with Camellia seed cake and after then mulched for 7 days before transplanting(pattern 3)was the best pattern compared to the other two patterns in which the soils were added with Camellia seed cake and then transplanted immediately(pattern 1)or the soils were added with Camellia seed cake and then stood for 7 days before transplanting(pattern 2).Addition of Camellia seed cake promoted the banana growth,improved the soil properties,increased the abundances of soil free-living nematode especially the bacterivores,and decreased the abundances of plant-parasites.Even more exciting,no plant-parasites were found in the treatments with 10 g/kg and 20 g/kg of Camellia seed cake.For the variations of the nematode indicaters,significant differences of H’ and J’ between the control and Camellia seed cake treatments were observed.The MI and PPI were decreased after addition of Camellia seed cake.NCR analysis showed ecology system of soil treated with Camellia seed cake was based on bacterial-based energy channel.In pattern 3,the disturbance of Camellia seed cake was lower than other two patterns.The soil microbial abundances and the soil catalase activity were increased after addition of the Camellia seed cake.The plant-parasite had significantly negative correlations with the bacteria abundance and an apart of fungi abundance while the bacterivores had significantly positive correlations with the bacteria abundance and the fungivores also had significantly positive correlations with fungi abundance;and the omnivorous had significant correlations with bacteria abundance and fungi abundance.5.Effect of Camellia seed cake on soil microfloraThe entire soil bacterial and fungal communities from pot experiment were analyzed using the MiSeq platform to investigate the effect of Camellia seed cake applied on the soil microflora.Based on 97%similarity,13,747 of bacterial OTUs were obtained from 2,014,903 sequence reads for 16S,while 2031 of fungal OTUs were obtained from 1,586,484 sequence reads for ITS.All sequence reads were derived from 39 separate soil samples and the numbers of high quality sequences per sample varied from 32,015 to 79,776 for bacteria and 22,579 to 56,473 for fungi.The coverage of all samples were higher than 0.91.The most abundant phylum of bacteria and fungi were Proteobacteria andAscomycota,respectively.Significant effects of Camellia seed cake application on soil microflora were observed.Compared to the soil samples collected from the control,Camellia seed cake-treated soil revealed higher abundances of Proteobacteria,Ascomycota,Basidiomycota and Zygomycota.Correlation analysis of soil microbial phylum relative abundance and soil nematode group showed Proteobacteria,Basidiomycota and Zygomycota had significant negative correlations with the plant-parasites.The most abundant genus of bacteria and fungi were Pseudomonas and Fusarium,respectively.Through Pearson correlation coefficients between the relative abundance of bacterial and fungal genus and soil nematode trophic groups,most of the nematode correlative bacterial and fungal genus were classified in Proteobacteria and Ascomycota,respectively.The Camellia seed cake treated soil revealed higher 15 bacterial and 7 fungal genus abundances,which showed significantly negative correlations with plant-parasites or positive correlations with soil free-living nematodes.We conjectured that Camellia seed cake could regulate soil microbial flora to ecological balance and soil health.6.The effect of Camellia seed cake in the field experimentField experiment results showed that Camellia seed cake application decreased the root-knot index by 76.7%in the bud stage and by 77.3%in the fruit maturity stage,while they were 18.0%in the bud stage and 71.8%in the fruit maturity stage for control.Soil pH,Ec,ammonium,nitrate,available phosphorus,and available potassium were increased after Camellia seed cake application.Camellia seed cake application significantly altered the soil nematode community,decreased the plant-parasites abundance,and increased the free-living nematodes abundance.A statistical analysis in the ecology index showed that application of Camellia seed cake increased soil nematode abundance and diversity and thus the ecological process was dominated by bacterial decomposition pathways.Moreover,Camellia seed cake application also significantly increased the bacteria abundance both in rhizosphere and bulk soil.Conclusion:Application of Camellia seed cake can significantly control banana root-knot especially when the application rate is 10 g/kg.This can be mainly attributed to the decrease of abundance and activities of M.javanica in soils,the provision of nutrients,and the improvements of communities both in soil nematodes and bacteria. |
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