Seedling Petri-dish inoculation method:A robust,easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops,leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen,R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here,we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use,takes about only 10 days from seed germination to the completion of inoculation assay,and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns,resistance of defense-related plant mutants,and virulence of R.solanacearum mutants.Furthermore,we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Drought adaptation of Bauhinia faberi var.Microphylla seedlings with dual inoculation of arbuscular mycorrhizal fungi

Bauhinia faberi via.Microphylla(BFM)is an important tree species for vegetation restoration in the dry valley of southwestern China.However,there were few studies on the application of arbuscular mycorrhizal fungi(AMF)in improving the drought adaptation of BFM.In order to investigate the response of BFM to water stress(WS),we tested four inoculation treatments((no AMF,Control),Glomus mosseae(GM),Glomus intraradices(GI),Glomus mosseae+Glomus intraradices(GMI))in pots,experimented under three field water holding capacity(WHC)of 70%,50%and 30%.The changes of seedling survival rate(SR),AMF relative root length colonization rate(Col),growth,photosynthetic parameters,water status and leaf nutrients were examined.The results showed that under 30%WHC drought conditions,SR with dual inoculation of AMF was not higher than with single inoculation of GM,suggesting that increasing the diversity of AMF did not definitely improve plant SR,and that the species of inoculated AMF might have an important impact on SR.The sensitivity of dual inoculated Col to water stress was lower than that of single inoculation,which was more favorable for dual inoculated BFM seedlings adapting to drought environment.The overall drought resistance ability(D)also showed that dual inoculation of AMF improved plant drought adaptation compared with single inoculation,which was related to the higher Col of dual inoculated AMF.This study is of practical importance to promote vegetation restoration in arid areas in a cost-effective and environmentally friendly manner.

Pathogenic Effects of Cloned Genomic DNA of Porcine Circovirus-like Virus P1 on Neonatal Mice via Different Inoculation Routes

[Objective] The paper was to explore the pathogenicity of cloned genomic DNA of porcine circovirus-like virus Pl to neonatal mice via different inoculation routes （brain, liver and muscle）. [Method] Cloned genomic DNA of P1 was inoculated to neonatal mice via different routes of brain, liver and muscle. Tissues of heart, liver, spleen, lung, kidney and brain were taken from neonatal mice at 7, 14 and 21 d post inoculation, re- spectively. Pl in various tissues were qualitatively and quantitatively detected by using ordinary PCR and quantitative real-time PCR. Meanwhile, histopathological changes were analyzed. [Result] Pl was detected in neonatal mice inoculated through three different routes. The viral load of tis- sues at 7 d post inoculation was significantly higher than those at 14 and 21 d post inoculation. Moreover, muscle inoculation led to the highest viral load in all tissues of neonatal mice. [Conclusion] Pl infection caused different degrees of pathological damage to heart, liver, lung, kidney and brain in neonatal mice.

Inoculation Effects of Dendrobium officinale Mycorrhizal Fungi on Their Plantlets

[Objective] This study aimed to explore the inoculation effects of Dendrobium officinale mycorrhizal fungi on their plantlets. [Method] Endophytic strains Tj1, Tj2 and Tj3 were obtained by isolation and purification from mycorrhiza of wild Dendrobium officinale and inoculated on the root system of Dendrobium officinale for inoculation test. [Result] Under tissue-culture conditions, at early stage, Tj1 strain hadn＇t shown promotion effect on Dendrobium officinale, Tj2 strain had shown relatively strong promotion effects, and Tj3 strain had promoted the growth of roots; at late stage, Tj1 strain had shown relatively strong promotion effects, Tj2 strain had shown the best inoculation effects and the strongest promotion effects, while Tj3 strain had caused root and seedling rot problems of the plantlets; under outdoor conditions, after inoculation with Tj2 strain, the number of leaves and lateral buds were increased, the growth of lateral root and the increase of plant height were significant, the leaves of Dendrobium officinale plantlets were large and dark green and an obvious root enlargement phenomenon was observed. [Conclusion] The two inoculation methods both indicate that Tj2 strain has relatively strong promotion effects on the growth of Dendrobium officinale roots and shoots, the increase of plant number and plant height, and the germination of new shoots and roots, which proved the effective establishment of symbiotic relationship between Tj2 strain and Dendrobium officinale. Therefore, T2 strain has practical application values on the successful cultivation of Dendrobium officinale plantlets.

Poisoning-free effect of calcium on grain refinement of Mg-3%Al alloy containing trace Fe by carbon inoculation

Mg-3%Al alloy was modified by combining Ca addition with carbon inoculation. The effects of Fe addition and addition sequence on the grain refinement were investigated. A higher grain refining efficiency could be obtained for the Mg-Al alloy modified by combining Ca addition with carbon inoculation. Fe addition and addition sequence had no obvious effect on the grain refinement. Ca addition could effectively avoid grain-coarsening resulting from Fe in the carbon-inoculated Mg-Al alloy. The Al-C-O particles, actually being Al4C3, should act as potent substrates for a-Mg grains in the sample treated by combining Ca addition with carbon inoculation. However, the duplex-phase particles of AI4C3 coated on Al-Fe or Al-C-Fe should be the potent substrates for a-Mg grains if Fe existed in the Mg-Al melt. Ca addition can contribute to the formation of the particles of Al4C3 coated on Al-Fe or Al-C-Fe, regardless of the Fe addition sequence. The poisoning effect of Fe was effectively inhibited in the carbon-inoculated of Mg-Al alloy due to Ca addition, namely, Ca has a poisoning-free effect.

Low-Density Co-Inoculation with Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 Promotes Plant Growth and Nitrogen Fixation in Soybean Cultivars

Inoculation density has a marked effect on nodulation and N fixation in soybean (Glycine max L.). Therefore, we conducted this study to determine the optimal inoculation density of Bradyhizobium japonicum SAY3-7 (SAY3-7) and Streptomyces griseoflavus P4 (P4) for plant growth, nodulation, and N fixation, and to investigate the effect of co-inoculation on selected soybean cultivars, using the optimal inoculation density. Nitrogen fixation, in terms of an acetylene reduction activity value, was measured using a flame ionization gas chromatograph. In this study, low-density single inoculation with P4 (105 or 106 cells mL-1) was associated with the highest plant biomass, compared with normal- and high-density single inoculation with P4 (107 or 108 cells mL-1). Moreover, low-density single or co-inoculations with SAY3-7 and/or P4 produced the highest nodule biomass and highest nitrogenase activity, compared with single or dual inoculation at other inoculation densities. Therefore, we evaluated low-density co-inoculation with P4 and SAY3-7, at the rate of 105 cells mL-1, on selected soybean cultivars. Low-density co-inoculation increased the plant biomass, compared with un-inoculated plants. The effects of single and co-inoculation on nodulation did not differ significantly for any of the cultivars, except “Yezin-9” in the first experiment and “Shan Seine” in the second experiment. Low-density inoculation with both bacteria increased N fixation by 15% - 75% for seven of the cultivars in the first experiment and by 15% - 39% for three of the cultivars in the second experiment, compared with single inoculation with SAY3-7. Based on the overall results, we concluded that low-density co-inoculation with P4 and SAY3-7 gave improved plant growth and N fixation.

Research of Synthetic Rules with Mechanical Properties vs Inoculation Degree on Gray Cast Iron

To fit the special needs of gray cast iron as a new engineering material,i.e.,higher σ b and lower HB,or lower σ b and higher HB,using the method of heat analyzing and polyparameters analytic figures,the synthetic rules of carbon saturation degree,inoculation degree and mechanical properties can be exposed.The result shows that,according to the needs of the part′s mechanical properties,referring to the synthetic rules of gray cast iron,the corresponding technological measure and composition proportion can be defined,by which we will get the exact mechanical properties that could achieve the result expected completely.Thus,the casting for gray cast iron might be 'designed'from'selected'.

Grain refinement of pure aluminum by direct current pulsed magnetic field and inoculation

The combined effects of direct current pulsed magnetic field （DC-PMF） and inoculation on pure aluminum were investigated, the grain refinement behavior of DC-PMF and inoculation was discussed. The experimental results indicate that the solidification micro structure of pure aluminum can be greatly refined under DC-PMF. Refinement of pure aluminum is attributed to electromagnetic undercooling and forced convection caused by DC-PMF. With single DC-PMF, the grain size in the equiaxed zone is uneven. However, under DC-PMF, by adding 0.05% （mass fraction） Al5Ti-B, the grain size of the sample is smaller, and the size distribution is more uniform than that of single DC-PMF. Furthermore, under the combination of DC-PMF and inoculation, with the increase of output current, the grain size is further reduced. When the output current increases to 100 A, the average grain size can decrease to 113 μn.

Fungal inoculation induces agarwood in young Aquilaria malaccensis trees in the nursery

Fungi are often used to induce agarwood inAquilariatrees. This study was conducted to evaluate the effect of several fungi on agarwood formation over time in youngAquilaria malaccensis （Lam.） trees. Typical changes in the length and light intensity of the resulting discoloration were observed after three and six month periods following inoculation. Wood samples were observed microscopically and classified into several light intensity groups. The discoloration length was meas-ured longitudinally. The duration after inoculation affected the mean of discoloration length： the 6-month old sample （1.70 cm） had a wider discoloration zone when compared to the 3-month old sample （1.17 cm）. When measuring the discoloration intensity, a positive relationship with time was perceived. Digital images, captured using a camera-equipped microscope, revealed that wood samples collected after six months appeared to be 1.8-times darker than after three months. We concluded that time, not the species of any of the tested fungi, had significant effect on discoloration length and intensity. Gas chromatography/mass spec-trometry （GCMS） analysis of the 6-month old sample yielded some important agarwood compounds such as benzylacetone, anisylacetone, guaiene and palustrol. This demonstrates that the tested fungi have the ability to induce agarwood formation in nurseryA. malaccensis trees.

Effect of inoculation with arbuscular mycorrhizal fungi on the degradation of DEHP in soil

The effect of inoculation with arbuscular mycorrhizal(AM) fungi( Acaulospora lavis ) on the degradation of di(2 ethylhexyl) phthalate(DEHP) in soil was studies. Cowpea plants (Pigna sinensis) were used as host plants and grown in a specially designed rhizobox. The experimental results indicated that, both in sterile and non sterile soil, mycorrhizal colonization rates were much higher in the mycorrhizal plants than in the non mycorrhizal plants. Addition of 4 mg/kg DEHP slightly affected mycorrhizal colonization, but the addition of 100 mg/kg DEHP significantly decreased mycorrhizal colonization. DEHP degradation in the mycorrhizosphere(Ms) and hyphosphere(Hs), especially in the Hs, increased after inoculation with Acaulospora lavis . It is concluded that mycorrhizal hyphae play an important role in the plant uptake, degradation and translocation of DEHP. The mechanism might be attributed to increased numbers of bacteria and actinomycetes and activity of dehydrogenase, urease and acid phosphatase in the Ms and Hs by mycorrhizal fungi.

A novel Al-10Si-2Fe master alloy and its ef ect on inoculation of eutectic cells in Sr-modii ed A356 alloy

To investigate the possibility of inoculating eutectic cells, a novel AI-10Si-2Fe master alloy was synthesized and tested in Sr-modified A356 alloy. The new master alloy that consists of reAl, Si andβ-AIsFeSi phases was prepared by a casting process, and the silicon phase was found to grow epitaxially from theβ-AIsFeSi particles. The inoculation efficiency of the new master alloy on Sr-modified A356 alloy has been investigated by quenching experiment and thermal analysis. With the addition of the new master alloy, the area density of eutectic cells is effectively increased by 100% and the eutectic growth temperature is increased by 1.5 ℃. Therefore, the new master alloy is deduced to introduce nucleating substrates for eutectic cells and to refine the eutectic cells in Sr-modifled A356 alloy. There is no poisonous interaction between the AI-10Si-2Fe master alloy and the Sr in this study. Consequently, the mechanical properties have been improved by the addition of the new master alloy.

Effect of ferrovanadium inoculation on microstructure and properties of high speed steel

The effect of ferrovanadium inoculation on the microstructure and properties of high speed steel (HSS) used for rolls was studied. The results showed that the as-cast eutectic carbide network tends to be broken after ferrovanadium inoculation, and the carbides are changed to rod-like or nodular shape. After heat treatment, the carbides in the inoculated HSS are spheroidized and distributed more uniformly in the matrix. The impact toughness of high speed steel with ferrovanadium inoculation is obviously improved. The action mechanism of ferrovanadium inoculation on the microstructure of the alloy is also discussed.

Effect of leachate recycling and inoculation on the biochemical characteristics of municipal refuse in landfill bioreactors

Activity development of key groups of enzymes involved in municipal refuse decomposition was measured in laboratory landfill bioreactors with and without leachate recycling and inoculation for about 210 days. The results showed that the enzymes (amylase, protease, cellulase, lipase and pectinase) were present in fresh refuse but at low values and positively affected by leachate recycling and refuse inoculation. The total average of cellulase activity in digesters D3 operated with leachate recycling but no inoculation, D4 and D5 operated with leachate recycling and inoculation was much higher than that in digesters D1 and D2 without leachate recycling and inoculation by 88%—127%, 117%—162% and 64%—98%. The total average of protease activity was higher in digester D4 than that in digesters D1, D2, D3 and D5 by 63%, 39%, 24% and 24%, respectively, and the positive effect of leachate recycling and inoculation on protease activity of landfilled refuse mainly was at the first two months. The total average of amylase activity was higher in digesters D3, D4 and D5 than that in digesters D1 and D2 by 83%—132%, 96%—148% and 81%—129%. During the early phase of incubation, the stimulatory effect of inoculation on lipase activity was measured, but refuse moisture was the main factor affecting lipase activity of landfilled refuse. The inoculation, initial and continuous inoculation of microorganisms existing in leachate, was the mainly stimulatory factor affecting pectinase activity of landfilled refuse.

Synergistic refining mechanism of Mg-3%Al alloy refining by carbon inoculation combining with Ca addition

Mg-3%Al alloy was refined by carbon inoculation combining with 0.2%Ca addition.High grain refining efficiency was obtained and the synergistic refining mechanism was deeply discussed in the present study.Al-C-O particles,actually Al4C3 particles,were formed in the carbon-inoculated Mg-3%A1 alloy acting as nuclei forα-Mg grains.Ca addition had no obvious effect on size distribution of the nucleating particles.Ca segregation was proved on Al4C3 particles,which should reduce the interfacial energy of nuclei/Mg.The constitutional undercooling in front of nucleus/liquid was increased from 0.12℃to 0.15℃induced by 0.2%Ca addition.The synergistic grain refining efficiency can be attributed to the higher constitutional undercooling and lower the interface energy of nucleus/Mg induced by Ca addition.More nucleating particles with small size could be activated acting as potent nuclei ofα-Mg grains.Consequently,Mg-3%Al alloy could be effectively refined due to the synergistic effect induced by carbon inoculation combining with Ca.

Early inoculation with caecal fermentation broth alters small intestine morphology,gene expression of tight junction proteins in the ileum,and the caecal metabolomic profiling of broilers

Background:The establishment of stable microbiota in early life is beneficial to the individual.Changes in the intestinal environment during early life play a crucial role in modulating the gut microbiota.Therefore,early intervention to change the intestinal environment can be regarded as a new regulation strategy for the growth and health of poultry.However,the effects of intestinal environmental changes on host physiology and metabolism are rarely reported.This study was conducted to investigate the effects of early inoculation with caecal fermentation broth on small intestine morphology,gene expression of tight junction proteins in the ileum,and cecum microbial metabolism of broilers.Results:Our data showed that early inoculation with caecal fermentation broth could improve intestine morphology.The small intestine villus height was significantly increased(P<0.05)in the intervened broilers compared to the control group,especially on day 28.A similar result was observed in the ratio of villus height to crypt depth(P<0.05).Meanwhile,we found early inoculation significantly increased(P<0.05)the expression levels of zonula occludens-1(ZO1)on days 14 and 28,claudin-1(CLDN1)on day 28,whereas the gene expression of claudin-2(CLDN2)was significantly decreased(P<0.05)on days 14 and 28.Gas chromatography time-of-flight/mass spectrometry(GC-TOF/MS)technology was further implemented to systematically evaluate the microbial metabolite profiles.Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)displayed a distinct trend towards separation between the fermentation broth group(F group)and the control group(C group).The differentially expressed metabolites were identified,and they were mainly functionally enriched in beta-alanine metabolism and biosynthesis of unsaturated fatty acids.In addition,1,3-diaminopropane was selected as a key biomarker that responded to early inoculation with caecal fermentation broth.Conclusions:These results provide insight into intestinal metabolomics and confirm that early inoculation with caecal fermentation broth can be used as a potential strategy to improve intestinal health of broilers.

Two stages kinetics of municipal solid waste inoculation composting processes

In order to understand the key mechanisms of the composting processes, the municipal solid waste(MSW) composting processes were divided into two stages, and the characteristics of typical experimental scenarios from the viewpoint of microbial kinetics was analyzed. Through experimentation with advanced composting reactor under controlled composting conditions, several equations were worked out to simulate the degradation rate of the substrate. The equations showed that the degradation rate was controlled by concentration of microbes in the first stage. The degradation rates of substrates of inoculation Run A, B, C and Control composting systems were 13 61 g/(kg·h), 13 08 g/(kg·h), 15 671 g/(kg·h), and 10 5 g/(kg·h), respectively. The value of Run C is around 1 5 times higher than that of Control system. The decomposition rate of the second stage is controlled by concentration of substrate. Although the organic matter degradation rates were similar to all Runs, inoculation could reduce the values of the half velocity coefficient K \-m and could be more efficient to make the composting stable. Particularly, for Run C, the degradation rate is high in the first stage, and K \-m is low in the second stage. The results indicated that the inoculation was efficient for the composting processes.

Inoculation with arbuscular mycorrhizal fungi improves seedlings growth of two sahelian date palm cultivars (<i>Phoenix dactylifera</i>L., cv. Nakhla hamra and cv. Tijib) under salinity stresses

This study presents an analysis of the impact of mycorrhizal inoculation on growth under salt stress of date palms cultivars Nakhla hamra (NHH) and Tijib known in the Sahel for their earliness in flowering and fruiting. The seedlings were grown in a greenhouse on a sandy substrate watered to field capacity every two days and were subjected to increasing levels of NaCl (0, 1, 2, 4, 6, 8 and 16 g·L-1) and then inoculated with 5 strains of Glomus (G. aggregatum, G. intraradices, G. verriculosum, G. mosseae, G. fasciculatum). The experimental design was a randomized complete block with three factors (cultivars: Tijib and Nakhla Hamra x seven levels of NaCl concentrations x six levels of inoculum). The results showed that in the absence of NaCl, G. verriculosum significantly enhanced shoot growth: 33.5 cm against 30.3 cm in the control and roots growth: 81.5 cm against 78 cm in NHH, while in Tijib, the growth is stimulated by Glomus fasciculatum: 33.8 cm against 32.7 cm for stems and 90 cm against 86 cm for the roots of inoculated plants and controls. In contrast, in the presence of NaCl 8 g·L-1, NHH has a better growth in the presence of G. intraradices: 37.3 cm against 30.6 cm for stems and 77 cm against 73 for roots, while Tijib grows better in the presence of G. fasciculatum with respectively 31.9 cm against 31.7 cm and 51.27 cm against 51.6 cm for stems and roots of inoculated plants and controls. Biochemical analysis revealed that changes in levels of proline depend on the cultivar, the mycorrhizal strain used and concentrations of NaCl applied. These results open the prospect of using mycorrhizal fungi to improve the productivity of palm trees in the Sahel.

Soybean Seed Co-Inoculation with <i>Bradyrhizobium</i>spp. and <i>Azospirillum brasilense</i>: A New Biotechnological Tool to Improve Yield and Sustainability

Legume nodulation by rhizobia can supply crops with nitrogen and reduce environmental impacts caused by chemical fertilization. The soybean crop in Brazil is an impressive example of how biological N2 fixation can be employed with a plant species of high economic value. However, the development of more productive cultivars, along with the increasing global climatic changes demand agricultural practices to become more productive and yet more environmentally friendly. Plant growth-promoting rhizobacteria (PGPR) are highly beneficial to agriculture worldwide, acting in plant nutrition, protection, and growth stimulation. Azospirillum is, certainly, the most employed PGPR in the world, but little is known about its interaction with rhizobia, when both are applied to legume seeds. We have evaluated the co-inoculation of bradyrhizobia and azospirilla on soybean seeds under different soil and climate conditions in Brazil. Our results demonstrated that co-inoculation is efficient and beneficial to the crop, and promotes yield increases without adding any chemical N fertilizers even in soils where established populations of soybean bradyrhizobia exist. The strategy of co-inoculation thus represents a new biotechnological tool to improve soybean yield without adding any chemical N fertilizers, thus contributing to current practices of sustainability in agriculture.

Effect of Inoculation with Effective Microorganisms and Leachate Recycle on Degradation of Municipal Refuse

Biodegradation of waste in landfill is a slow process requiring decades for completion. Accelerated degradation of municipal refuse in modulated landfill environments may alleviate or eliminate pollution to the land, water and air. In this work, nineteen effective microorganisms (EMs) were isolated from old landfill refuse by enrichment culturing techniques and used for the inoculum of municipal refuse. The preliminary experiments demonstrate that a combination of EMs inoculation in landfill with leachate recycle resulted in increased rates of decomposition and faster process stability. The concentrations of COD, VFA and SO4^2- in digester with EMs inoculation and leachate recycle decreased more rapidly than others. Gas production from digester with EMs inoculation and leachate recycle commenced around 32 days, which is a week shorter than with leachate recycle only. And peak cumulative gas production was obtained much earlier in digester with EMs inoculation and leachate recycle (150 days) compared to 180 days with leachate recycle only. Moreover, in the first two months, the rate of settlement in digester with EMs inoculation and leachate recycle was more rapid than others.

Breeding for FHB Resistance via Fusarium Damaged Kernels and Deoxynivalenol Accumulation as Well as Inoculation Methods in Winter Wheat

FHB is one of the most destructive diseases of wheat. Resistance testing depends strongly on inoculation methods, and on measured traits. Therefore a four-year (2009-2012) study was performed using spray inoculation + polyethylene (PE) bag cover, spray inoculation + mist irrigation, and spawn method supported by mist irrigation on 40 genotypes, 20 from Hungary and 20 from IFA Tulln, Austria. Each year four isolates were used in artificial inoculations except the spawn method where stalk debris served the inoculum. Visual Fusarium head blight (FHB) scores, Fusarium damaged kernels (FDK) and deoxynivalenol (DON) contamination were checked. 7680 FHB and FDK, as well as 3840 DON analyses served as the background for the statistical evaluation. The most reliable method used was the spray + polyethylene (PE) bag;the other two were significantly poorer being valid for all traits. The FHB scores were the least reliable, whereas the FDK was much more consequent and the DON gave the best results. The FDK gave much better predictions for DON contamination than FHB. The cultivars responses correlated well at different epidemic severities. The presence of the kernel resistance was confirmed and a new trait as extra kernel susceptibility was described. Presence of DON resistance was confirmed again, and extra DON susceptibility was described as a new trait. DON performance varied on the most sensitive cultivar between 0.32 and 143 mg/kg (mean 17.52 mg/kg) and on the most resistant genotype between 0.00 and 18.19 mg/kg (mean 1.87 mg/kg). Correlations between stability and resistance level are r = 0.85 for FHB, 0.78 for FDK, and 0.88 for DON, all at a significance level of p = 0.001. The very close correlation between FDK and DON contamination (r = 0.81, p = 0.001) proves that control of DON contamination needs appropriate resistance. In the breeding program evaluation of FDK is the most important, and then DON will be decided. Variety registration must be updated;otherwise no improvement on the field will occur.