Biological soil crusts and their potential applications in the sand land over Qinghai-Tibet Plateau

The Qinghai-Tibet Plateau is now experiencing ecological degradation risks as a result of climate change and human activities.The alpine grassland ecology in permafrost zones is fragile and susceptible to deterioration due to its high altitude,low temperature,and limited oxygen,which complicates the repair of damaged land.Biological soil crusts(BSCs)are crucial for land restoration in plateau regions because they can thrive in harsh conditions and have environmentally beneficial traits.Inoculated biological soil crust(IBSC)has shown success in low-altitude desert regions,but may not be easily duplicated to the plateau environment.Therefore,it is essential to do a comprehensive and multifaceted analysis of the basic theoretical comprehension and practical application of BSCs on the Tibetan Plateau.This review article aims to provide a brief summary of the ecological significance and the mechanisms related to the creation,growth,and progression of BSCs.It discusses the techniques used for cultivating BSCs in laboratories and using them in the field,focusing on the Qinghai-Tibet Plateau circumstance.We thoroughly discussed the potential and the required paths for further studies.This study may be used as a basis for selecting suitable microbial strains and accompanying supplemental actions for implementing IBSCs in the Qinghai-Tibet Plateau.

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.

Biotransformation of Shrimp Wastes by Bacillus subtilis OKF04 and Evaluation of Growth Promoting Effect in Crop Planting

In this study,we proposed a reliable and sustainable technique for the clean utilization of shrimp wastes,which can yield a solid inoculant of Bacillus subtilis OKF04 containing micronutrients at low cost without the risk of contamination.Study of the culture conditions revealed that the head of shrimp Litopenaus vannamei and the wheat bran acted as suitable substrates for the growth of B.subtilis OKF04.With 60%initial moisture content,30℃culture temperature,and 5%inoculation amount,followed by 48 hours of fermentation and 0.5%soluble starch added during the drying process(50℃for 6h),a solid B.subtilis OKF04 inoculant with a spore amount of 2.4×10^(10)CFU g^(-1)and a high amino acid content was obtained.The solid B.subtilis OKF04 inoculant was applied to cultivate pakchoi under pot experiment.As the result,of adding to,the size of stems and leaves,nutritional composition,and physiological activity of pakchoi were significantly(P<0.05)enhanced by solid B.subtilis OKF04 inoculant.B.subtilis OKF04 also significantly(P<0.05)increased the soil’s nutrient content and improved its microbial composition.Furthermore,pakchoi cultivated with a low dose of solid B.subtilis OKF04 inoculant(0.05 g kg^(-1)soil)resulted in the best results.This study provides a new method for the preparation of microbial inoculants with solid waste shrimp heads.

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.

Establishment and Application of Identification Methods for Resistance to Sugarcane White Leaf Disease(SCWL)

[Objectives]This study was conducted to establish simple, efficient, stable, standardized and practical identification methods for sugarcane resistance to white leaf disease(SCWL), and promote the breeding for sugarcane resistance to SCWL. [Methods]The identification technology of sugarcane resistance to SCWL was systematically studied and explored from the aspects of sugarcane material treatment and planting, inoculation liquid preparation, inoculation method, disease investigation, grading standard formulation, etc., and two sets of simple, efficient, stable, standardized and practical accurate identification methods for sugarcane resistance to SCWL were created for the first time, namely, the seed cane coating inoculation method and the stem-cutting inoculation method at the growth stage. The seed cane coating inoculation method includes the steps of directly screening SCWL phytoplasma, extracting juice from cane and adding 10 times of sterile water to prepare an inoculation liquid, spraying seed cane on plastic film to keep moisture, planting the inoculated materials in barrels in an insect-proof greenhouse for cultivation, investigating the incidence rate 30 d after inoculation, and evaluating the disease resistance according to the 1-5 level standard. The method of stem-cutting inoculation includes the steps of directly screening sugarcane stems carrying SCWL phytoplasma and adding 10 times of sterile water to prepare an inoculation liquid, cultivating the identification materials in an insect-proof greenhouse, dropping 100 μl of the inoculation liquid into each root incision with a pipette gun at the age of 6 months, investigating the incidence rate 20 d after planting, and evaluating the disease resistance according to the 1-5 level standard. [Results] The two methods are similar to the natural transmission method. After inoculation, SCML occurred significantly, with high sensitivity and good reproducibility. The results of resistance identification were consistent with those of natural disease in the field. Through the two inoculation methods and field natural disease investigation, the resistance of 10 main cultivars to SCML was identified, which was true and reliable. [Conclusions] This study can provide standard varieties for identification of SCML resistance in the future.

Recurred forehead osteoma disseminated after previous osteoma excision:A case report

BACKGROUND Forehead osteoma is a commonly encountered benign facial bone tumor.Endoscopic excision of benign forehead masses is widely performed.Here,we report a rare case of recurrent forehead osteoma that disseminated after a previous osteoma excision.CASE SUMMARY A 54-year-old female patient had previously undergone endoscopic removal of a single forehead osteoma at 30 years of age.However,she had a recurrent osteoma around the same site and underwent another endoscopic resection at 40 years of age.During her first visit to our outpatient clinic,she presented with a cobblestone-like irregular surface on the forehead and a 3D facial bone computed tomography scan revealed a widely ragged surface of the inoculated osteoma on the outer table of the frontal bone.Under general anesthesia,we performed a radical complete excision of the disseminated osteoma through a bicoronal incision using an osteotome,chisel,mallet,and rasping.We hypothesized that the recurrence may have been caused by the inoculation of residual osteoma remnants from the previous procedure.Craniofacial surgeons should be cautious when removing osteoma particles,particularly when using an endoscopic approach.CONCLUSION To prevent recurrence,it is essential to conduct additional meticulous burring and a thorough inspection of the surface after copious irrigation.

Tissue Culture Application of Maranta arundinacea

The reproductive methods of ornamental arrowroot(Maranta arundinacea)mainly include plant division,cutting propagation,truncation and sprout promotion propagation,and tissue culture.Large-scale seedling production mainly relies on tissue culture.In the process of tissue culture,the culture conditions can be artificially controlled,and the plant materials used for tissue culture grow completely in artificially-provided culture media and microclimate environment,free from the adverse effects of catastrophic climate,seasonal and diurnal changes.Because the conditions are relatively uniform,tissue culture is very beneficial for plant growth and more convenient for stable long-term cultivation and production.In the process of tissue culture,the inoculation process is more important.How can we achieve standardized operation and reduce pollution during the inoculation process is worth exploring.

Culture Media Options for Growth and Morphological Characterisation of Cercospora coffeicola Affecting Coffee in Zimbabwe

Cercospora leaf spot is fast turning into a critically important disease in Zimbabwe.The disease is caused by Cercospora coffeicola which significantly reduces productivity and quality of coffee.Disturbingly,optimum sporulation of Cercospora coffeicola in culture remains a limiting factor for microbial analysis and quantitative studies of Cercospora leaf spot.Faced with this challenge,an in-vitro study was conducted at Coffee Research Institute,Manicaland,Zimbabwe to examine growth of Cercospora coffeicola in different nutrient media and to determine the best media for Cercospora coffeicola analysis.Six nutrient media were assessed(corn meal agar,oat meal agar,Czapek Dox agar,malt extract agar,yeast extract agar and potato dextrose agar)for the growth of Cercospora coffeicola.The laboratory-based experiment was duplicated,laid out in a Completely Randomized Design,replicated three times and based on Cercospora coffeicola nutrient inoculation.Data were collected on radial growth,colour and texture of mycelium at 3 and 6 days after inoculation.There were significant differences(p<0.05)in the growth of Cercospora coffeicola in media after 3 and 6 days.Malt extract agar had the greatest radial growth(34 mm and 32 mm)of Cercospora coffeicola for trials 1 and 2 respectively,whilst the least growth was in the oat meal agar(14.2 mm and 15.7 mm)for trials 1 and 2 respectively.There were variations in colour and texture of mycelium with malt extract agar,potato dextrose agar and oat meal agar associated with darker colours and rough texture while smooth white mycelia were found in corn meal agar.After considering all nutrient media,malt extract agar was found to be the best media for the growth of Cercospora coffeicola in-vitro.On the basis of our findings,the authors recommend the use of malt extract agar as the primary media for identification and characterisation of Cercospora coffeicola.

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.

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.

Transient Expression of BYDV-MP in Nicotiana benthamiana

[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.

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.

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'.

骨诱导天然煅烧骨无菌检查方法学验证

目的:建立骨诱导天然煅烧骨无菌检查方法,保证检验结果的准确性和可靠性。方法:按《中国药典》2010年版三部(附录ⅦA)无菌检查法中的直接接种法进行。结果:样品管无菌生长,六株阳性对照菌生长良好。结论:方法学验证采用直接接种法进行的无菌检查,可行。

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 Bacillus amyloliquefaciens and Bacillus subtilis on fermentation, dynamics of bacterial community and their functional shifts of whole-plant corn silage

Background:Bacillus amyloliquefaciens(BA)and Bacillus subtilis(BS)are usually used as feed supplements directly or bacterial inoculants in biological feeds for animals.However,few research have reported the effects of BA and BS on fermentation characteristics and bacterial community successions of whole-plant corn silage during ensiling.If the BA and BS inoculants have positive effects on silages,then they could not only improve fermentation characteristics,but also deliver BA or BS viable cells to ruminants,which would play its probiotic effect.Therefore,the objectives of this study were to investigate the effects of BA and BS on the fermentation,chemical characteristics,bacterial community and their metabolic pathway of whole-plant corn silage.Results:Freshly chopped whole-plant corn was inoculated without or with BA and BS,respectively,and ensiled for1,3,7,14 and 60 d.Results showed that BA and BS inoculations increased lactic acid concentrations of whole-plant corn silages compared with control,and BA inoculation decreased acetic acid concentrations,whereas BS inoculation decreased fiber contents and increased crude protein(CP)content.Higher water-soluble carbohydrate contents and lower starch contents were observed in BA-and BS-inoculated silages compared with that in control.The decreased CP content and increased non-protein nitrogen content were observed in BA-inoculated silage,which was consistent with the higher amino acid metabolism abundances observed in BA-inoculated silage.In addition,it was noteworthy that BA and BS inoculations increased the metabolism of cofactors and vitamins,and decreased the relative abundances of drug resistance:antimicrobial pathways.We also found that the bacterial metabolism pathways were clearly separated into three clusters based on the ensiling times of whole-plant corn silage in the present study.There were no significant differences in bacterial community compositions among the three groups during ensiling.However,BA and BS inoculations decreased the relative abundances of undesirable bacteria such as Acetobacter and Acinetobacter.Conclusion:Our findings suggested that the BS strain was more suitable as silage inoculants than the BA strain in whole-plant corn silage in this study.

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.

Grain refinement of Mg-Al alloys inoculated by MgAl_(2)O_(4)powder

As a potent nucleating substrate forα-Mg grain,MgAl_(2)O_(4) powder was used to inoculate the Mg-Al melt in this study.The effects of MgAl_(2)O_(4)amount,holding time and Al content on the grain size and grain refining ratio of the inoculated Mg-Al alloys are systematically investigated.The results show that the minimum grain size of Mg-3Al alloy is achieved by adding 2wt.%MgAl_(2)O_(4)powder and this alloy exhibits higher grain refining ratio than Mg-5Al and Mg-8Al alloys.The crystallographic misfit calculation indicates the wellmatching and possible orientation relationships(ORs)betweenα-Mg and MgAl_(2)O_(4).Among these predicted ORs,[10–10]α−Mg//[110]MgAl2O4 in(0002)α−Mg//(1–13)MgAl2O4 possesses the smallest misfit,i.e.,2.34%(fr).Both results of the experiment and crystallographic calculation demonstrate that the grain refinement of Mg-Al alloys is attributed to the MgAl_(2)O_(4)particles acting as the heterogeneous nucleation substrates forα-Mg grains.

Use of two fungal species to induce agarwood resin formation in Gyrinops walla

Gyrinops walla Gaertn. is the only species growing in Sri Lanka that belongs to the agarwood family,Thymelaeaceae. Although agarwood resin induction and extraction from Aquilaria species of the same family have been practised for many decades in Southeast Asian region,the ability of producing agarwood resins in G. walla was discovered recently. Since previous studies were on agarwood resins formed due to natural causes, the present study was conducted to identify the potential fungal species that are capable of artificially inducing agarwood resin formation in G. walla. Since this is the first ever study conducted on artificial inducement of agarwood resin formation in G.walla, Aspergillus niger and Fusarium solani were selected owing to their high abundance in the naturally formed agarwood resinous tissues collected from 25 G. walla trees.Both fungal species were separately grown in yeast extract glucose agar and used to inoculate healthy G. walla trees under aseptic conditions. Three holes were made for each tree and 2 g of fungal culture including the medium were placed in each hole. Tissue discoloration, characteristic aroma, resin content and resin constituents were checked at10 cm intervals above and below the inoculation points for a period of 1 year. Results revealed that tissue discoloration and resin content were higher in the trees inoculated with A. niger. Other than at 10 cm above and below the inoculation points, samples collected at all locations had significantly higher resin contents when inoculated with A. niger compared to F. solani. Sixteen agarwood resin constituents, which were also recorded in Aquilaria species, were identified from the discolored tissues using GC–MS analysis. Jinkohol, agarospirol and 2(2-phenyl)chromone derivatives were found in all discolored tissues collected at 10-cm intervals of the trees inoculated with both fungi. b-Seline, c-eudesmol and valerenal were found in nine of 10 sample points on the stem. c-Elemene was recorded only in one sample. The characteristic aroma during burning was stronger for dark-colored tissues than the light-colored ones. The present study confirmed the potential use of certain fungal species to induce agarwood resin in G. walla and that A. niger is more effective than F.solani.

Nodule Formation and Development in Soybeans(Glycine max L.) in Response to Phosphorus Supply in Solution Culture

Phosphorus (P) is necessary for growth and nitrogen fixation, and thus its deficiency is a major factor limiting legume production in most agricultural soils. The effect of phosphorus supply on nodule development and its role in soybeans (Glycine max L.) was studied in a nutrient solution. Plants were inoculated with Bradyrhizobium japonicum and grown for 35 days in a glasshouse at a day and night temperature of 25℃and 15℃, respectively. Although increasing P supply increased the concentrations of P and N in the shoots and roots, the external P supply did not significantly affect the P concentration in the nodules, and the N fixed per unit nodule biomass decreased with increasing P supply. The nitrogen content in the shoots correlated well with the P content (r = 0.92**). At an inoculation level of 102 cells mL-1, the P supply did not affect the number of nodules; however, at inoculation levels of 103.5 and 105 cells mL-1, increasing P supply increased both the number and size of nodules. Irrespective of the inoculation level, increasing P supply increased the nodule biomass relative to the biomass of the host plant. It is suggested that the P deficiency specifically inhibited the nodule development and thereby the total N2 fixation.