THE INFLUENCE OF PENICILLIUM FUNGI ISOLATED FROM SOYBEAN ROOTS ON THE SYMBIOTIC SYSTEM “GLYCINE MAX – BRADYRHIZOBIUM JAPONICUM” AND CROP PRODUCTIVITY

Two-field experiments were conducted at Nubaria Agricultural Research Station, ARC. in summer seasons of 2006 and 2007; consecutively to study the response of soybean cultivar Giza 111, inoculated with Bradyrhizobium japonicum, to different rates of phosphorus fertilizer in the presence of Bacillus megatherium, Bacillus polymyxa or a mixture of both to give a highlight on the role of these microorganisms in increasing enzymatic activity in soil after 75 days of sowing and in increasing soybean yield and N, P and K seeds contents under the calcareous soil conditions in Nubaria. With respect to nitrogenase activity of nodules and rhizosphere and nodules weight, data show that the inoculation increased significantly nitrogenase activity (N2- ase) of nodules in both seasons, while it gave a significant increase in nodules weight of the 2nd season. N2- ase of rhizosphere soil increased significantly in the 1st season as a result of inoculation. Regarding the effect of inoculation and phosphorus fertilizer on dehydrogenase activity (DHA) of rhizosphere soil, results indicated that inoculation with both bacteria led to a significant increase in DHA only in the 2nd season. Results showed also that inoculation of soybean seeds with a mixture of both bacteria plus 30 kg P2O5/fed gave the highest significant grain yield, 100-grain weight, protein yield and N, P, and K contents of soybean seeds in both tested seasons over uninoculated treatments. Consequently, results revealed that inoculation of soybean seeds with B. polymyxa combined with 30 kg P2O5/fed produced significantly the highest oil yield as compared to uninoculated treatment. It can be concluded that the improvement of soybean yield and its quality could be achieved by the application of 30 kg P2O5/fed together with inoculation of seeds with (Bradyrhizobium japonicum and a mixture of B. megatherium, B. polymyxa) under calcareous soil conditions.

Regulation of nitrogenase activity in soybean nodules by ATP and energy charge

The aim of this study was to investigated the biological diversity, antibacterial activites and the plant growth-promoting traits of endophytic fungi of sandal (Santalum album), and to assess their potential in the development of antibacterial substances and rapid cultivation of sandal. The results of isolation and taxa analysis of endophytic fungi from sandal showed that 325 strains of endophytic fungi belonging to 16 genera of endophytic fungi were isolated from sandal (of which 86 from roots, 105 from stems and 134 from leaves). The isolation rate and colonization rate of endophytic fungi in different sandal parts showed the same pattern of change: leave>stems>roots. The diversity index of endophytic fungi in sandal roots was significantly higher than that of stems and leaves. The dominant endophytic fungi of sandal roots, stems and leaves showed significant differences. The dominant endophytic fungi of roots were Fusarium (50.00%) and Alternaria (10.47%), Alternaria (58.11%) and Acremonium (20.00%) for stems, and Pantoea (74.63%) for leaves. The antibacterial activity of 40 representative strains of sandal endophytic fungi were analyzed and the results showed that 90% of endophytic fungi exhibited inhibitory activity against at least one of the tested bacteria strains, and the strains with inhibitory activity to Escherichia coli, Enterobacter aerogenes, Shigella dysenteriae, Salmonella typhimurium, Staphylococcus aureus, and Bacillus subtilis accounted for 45.0%, 30%, 47.5%, 55%, 72.5%, and 62.5%, respectively. The sandal fungal endophytes with plant growth-promoting characteristics were screened, and 5 strains of endophytic fungi with phosphorus-solubilizing activity, 8 strains of endophytic fungi producing IAA, and 4 strains of endophytic fungi producing siderophores were found. Among them, endophytic fungus Monilia sp TXRF45 clould produced IAA and siderophores, and also show phosphate-solubilizing activity. The results indicated that the endophytic fungi of Sandal were rich in species diversity and their distribution had a certain tissue specificity. Some strains showed good antibacterial activity and growth-promoting properties, which could potentially applicable for the development of antibacterial substances and rapid cultivation of sandal.

<p id="C3">Plant hormone auxin plays a vital role in the growth and development of plants. Auxin homeostasis and concentration gradient establishment control the polar formation of almost all organs. The synthesis, transportation, perception, and metabolic degradation of auxin in specific cells establish a concentration gradient of auxin in accordance with organ development. In legumes, roots interact with soil microorganisms to form a special organ called nodules, which is used for biological nitrogen fixation. However, the function of auxin homeostasis control of biological nitrogen fixation is unknown. Studies showed that PIN-Like (PILS) proteins in<italic> Arabidopsis</italic> helped to regulate intracellular auxin homeostasis and mediate auxin signal transmission in the downstream nucleus. In this study, 19 PILS family genes (<italic>GmPILSs</italic>) were identified in soybean genome and distributed unevenly on 10 chromosomes of soybean. <italic>GmPILSs</italic> exhibited a variety of expression patterns in nine tissue parts of soybean, and had obvious specificity of tissue expression. <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> were enriched and expressed in the rhizobia region, and the expression of <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> in nodules was down-regulated by artificial microRNA interference (amiRNAi), resulting in the increase of nitrogenase activity in the nodules. However, the overexpression of <italic>GmPILS1f</italic> leaded to the decrease nitrogenase activity in root nodules, <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> might participate in the regulation of soybean nitrogenase activity. These results lay the foundation for further analysis of the function and mechanism of soybean <italic>GmPILS</italic> family genes, and also provide valuable genetic resources for the application of nodulation and nitrogen fixation in agricultural breeding.

Preliminary studies have indicated that after addition of C2H2 there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata. The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C2H4 and CO2 evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C2H2. The peak rate of C2H4 evolution occurred at 30 s after C2H2 exposure, while the rate of CO2 evolution started to decline at 60 s after exposure to C2H2. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO2 evolution. Further, pretreatment with Ar eliminated most of the C2H2‐induced decline in nitrogenase activity and CO2 evolution. These C2H2‐ and Ar‐induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.

We studied the effect of complex seed treatment with fungicides and rhizobium culture on the activity of phenolic metabolism enzymes – polyphenol oxidase and guaiacol peroxidase in the early stages of the formation and functioning of various symbiotic systems Glycine max – Bradyrhizobium japonicum. In the research we used microbiological, physiological, biochemical methods, gas chromatography and spectrophotometry. The objects of the study were selected symbiotic systems formed with the participation of soybean (Glycine max (L.) Merr.), Diamond variety, strains Bradyrhizobium japonicum 634b (active, virulent) and 604k (inactive, highly virulent) and fungicides Maxim XL 035 PS (fludioxonil, 25 g/L, metalaxyl, 10 g/L), and Standak Top (fipronil, 250 g/L, thiophanate methyl, 225 g/L, piraclostrobin, 25 g/L). Before sowing, the seeds of soybean were treated with solutions of fungicides, calculated on the basis of one rate of expenditure of the active substance of each preparation indicated by the producer per ton of seed. One part of the seeds treated with fungicides was inoculated with rhizobium culture for 1 h (the titre of bacteria was 108 cells in 1 ml). The other part of the fungicide-treated seeds was not inoculated by rhizobium culture. As a result of the research, it was revealed that an effective symbiotic system formed with the participation of soybean plants and the active strain rhizobia 634b is characterized by a high level of polyphenol oxidase activity and low guaiacol peroxidase in roots and root nodules in the stages of second and third true leaves. Such changes in the activity of enzymes occurred along with the formation of nodules which actively fixed the molecular nitrogen of the atmosphere. An ineffective symbiotic system (strain 604k) is characterized by an elevated level of polyphenol oxidase activity in the roots and guaiacol peroxidase in the root nodules, which is accompanied by activation of the process of nodulation. Treatment of soybean seeds with fungicides in an effective symbiotic system leads to a change in the activity of the enzymes of the phenolic metabolism, which induced adaptive changes in plant metabolism and growth of nitrogenase activity of the root nodules. The recorded changes in the activity of both enzymes for the action of fungicides in the ineffective symbiotic system can be considered as a kind of response of the plant to the treatment and were observed along with the reduction of the processes of nodulation into the stage of the third true leaf.

Symbiotic systems created by soybean variety Almaz (Glycine max (L.) Merr.) and strains of nodule bacteria Bradyrhizobium japonicum 646, РС09, В157 with were investigated under different water supply are investigated (30 and 60 % field capacity, FC). It was shown that drought inhibited the process of nodulation, depending on the functional and adaptive capacity of each of the microsymbionts. The number and mass of root nodules on plants decreased compared to control plants under insufficient watering. Insufficient watering (30 % FC) negatively impacted the functioning of symbiotic systems Glycine max (L.) Merr.—Bradyrhizobium japonicum depending on the drought duration. Strains of Bradyrhizobium japonicum PC09 and B157 formed more drought-tolerant symbiotic systems. They had higher nitrogen fixing activity under reduced moisture supply (30 % FC) and during the recovery period compared to the Bradyrhizobium japonicum 646 strain. It was shown that with increasing drought duration (from the 3rd to the 10th day) the difference in physiological and symbiotic parameters between treated and control plants increased, but with the resumption of watering they partially leveled depending on the inoculant strain.

Soybean (Glycine max (L.) Merrill) is one of the most important crops worldwide providing dietary protein and vegetable oil. Most of the nitrogen required by the crop is supplied through biological N2 fixation. Non-thermal plasma is a fast, economical, and environmental-friendly technology that can improve seed quality, plant growth, and crop yield. Soybean seeds were exposed to a dielectric barrier discharge plasma operating at atmospheric pressure air with superimposed flows of O2 or N2 as carrying gases. An arrangement of a thin phenolic sheet covered by polyester films was employed as an insulating barrier. We focused on the ability of plasma to improve soybean nodulation and biological nitrogen fixation. The total number of nodules and their weight were significantly higher in plants grown from treated seeds than in control. Plasma treatments incremented 1.6 fold the nitrogenase activity in nodules, while leghaemoglobin content was increased two times, indicating that nodules were fixing nitrogen more actively than control. Accordingly, the nitrogen content in nodules and the aerial part of plants increased by 64% and 23%, respectively. Our results were supported by biometrical parameters. The results suggested that different mechanisms are involved in soybean nodulation improvement. Therefore, the root contents of isoflavonoids, glutathione, auxin and cytokinin, and expansin (GmEXP1) gene expression were determined. We consider this emerging technology is a suitable pre-sowing seed treatment.

The legume Dalbergia sissoo Roxb is an important forest tree of the arid and semi-arid regions. The efficiency of Bradyrhizobium (Ds Rhz-9) and Glomus fasciculatum (Thaxter sensu Gerd.) Gerd. and Trappe to promote growth of D. sissoo and the effect of indole acetic acid (IAA) on the tripartite symbiotic association was studied in a pot experiment. Parameters like plant growth, nodulation, total phosphorus, total nitrogen content, nitrogenase activity of root nodules (acetylene reduction assay), and arbuscular mycorrhizal fungi (AMF) root colonization were analyzed. The dual inoculation of Bradyrhizobium along with G. fasciculatum showed synergistic increase in growth, nodulation, and nitrogen fixation of the seedlings. The maximum nitrogen (1.45%) and phosphorus (34.6%) content of dry weight was recorded in the dual inoculated plants when treated with IAA (1.39 × 10−3 mole/liter). The nitrogenase activity of the nodules in dual treatments showed an enhancement of 34.9%, which became further enhanced by 15.7% when treated with IAA. The presence of Bradyrhizobium in the rhizosphere was found to decrease AMF root colonization. In dual inoculated plants, the exogenous application of IAA (1.11 × 10−3 moles/liter) increased AMF root colonization by 60%. The tripartite synergistic interaction of Bradyrhizobium and G. fasciculatum with D. sissoo seems to be promising and can be utilized for its growth promotion in forest nurseries and reforestation of degraded areas. Exogenous application of IAA will be beneficial and recommended.

Phenylalanine ammonia-lyase (PAL) is a key enzyme of the phenylpropanoid pathway and provides precursors for the synthesis of many secondary metabolites, which are necessary for the development and protection of plants from external factors of various natures, in particular plays an important role in the formation and development of their symbiosis with microorganisms. Aim. To study the activity of PAL in soybean plants in the early stages of legume-rhizobial symbiosis under the influence of seed inoculation with Bradyrhizobium japonicum strains with different symbiotic properties on the background of fungicide treatment. Methods. Microbiology (bacterial culture growing, seeds inoculation), physiological (pot experiment), biochemical (determining the PAL enzyme activity). Results. Inoculation of soybean seeds with active virulent rhizobia induces a significant decrease in PAL activity in the roots at the primordial leaf stage and a significant increase in its activity level at the first true leaf stage, compared to inactive symbiosis. At the stage of third true leaf, the activity of PAL increased more significantly in soybean root nodules formed by inactive rhizobia, compared to active symbiosis. However, at the stage of third true leaves, the activity of PAL in soybean root nodules formed by inactive rhizobia increased significantly compared to active symbiosis. The use of fungicides for pre-sowing treatment of soybean seeds induces changes in the level of PAL activity in roots and nodules, which do not affect the overall dynamics of enzyme activity in different effective symbiotic systems Glycine max - Bradyrhizobium japonicum. Conclusions. The activity of PAL in the roots and especially in the root nodules of soybeans in the early stage of plant development in the case of fungicides using and bacterization is primarily due to the action of the inoculation factor, and is determined by the symbiotic properties of rhizobia strains, in particular, their virulence and nitrogen fixation activity.

N2 fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of “biological” nitrogen in plants as determined by the 15N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N2 fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.

A inoculação com bactérias do gênero Bradyrhizobium, ocasiona na rizosfera a formação estruturas especializadas na fixação biológica de nitrogênio, sendo descartada a aplicação na cultura da soja, uma vez que a relação simbiótica consegue suprir suas necessidades. O objetivo do trabalho foi avaliar a influência da inoculação bem como de níveis de nitrogênio em cobertura no crescimento, desenvolvimento e produtividade da soja. O experimento foi realizado na área experimental da Universidade Estadual de Mato Grosso do Sul – Unidade Universitária de Aquidauana. O delineamento experimental foi em blocos casualizados em esquema fatorial 4x4, com quatro repetições. Os tratamentos foram constituídos pela combinação entre inoculantes (Bradyrhizobium japonicum; Azospirillum brasilense; B. japonicum + A. brasilense; Controle sem inoculação) e quatro níveis de nitrogênio em cobertura na soja (0; 50; 100; 150 kg ha-1). A inoculação foi realizada antes da semeadura, já a aplicação do nitrogênio foi realizada no estádio V8, para tal, utilizou-se sulfato de amônio como fonte. Foram avaliados: população final de plantas, altura da planta, altura de inserção da primeira vagem, diâmetro do caule, número de vagens por planta, número de grãos por vagem, produtividade de grãos, massa de 1000 grãos. A inoculação de sementes de soja usando isoladamente ou em conjunto não interfere no desenvolvimento e produtividade de grãos da cultura da soja, assim o uso de nitrogênio mineral em cobertura é dispensável, pois não reflete em incrementos na produtividade de grãos da soja.

In the context of climate change and human factors, the drought problem is a particularly serious one, and environmental pollution caused by the abuse of chemical fertilizers and pesticides is increasingly serious. Endophytic fungi can be used as a protection option, which is ecologically friendly, to alleviate abiotic stresses on plants, promote plant growth, and promote the sustainable development of agriculture and forestry. Therefore, it is of great significance to screen and isolate endophytic fungi that are beneficial to crops from plants in special habitats. In this study, endophytic fungi were isolated from Cotoneaster multiflorus, and drought-tolerant endophytic fungi were screened by simulating drought stress with different concentrations of PEG-6000, and the growth-promoting effects of these drought-tolerant strains were evaluated. A total of 113 strains of endophytic fungi were isolated and purified from different tissues of C. multiflorus. After simulated drought stress, 25 endophytic fungi showed strong drought tolerance. After ITS sequence identification, they belonged to 7 genera and 12 species, including Aspergillus, Fusarium, Colletotrichum, Penicillium, Diaporthe, Geotrichum, and Metarhizium. According to the identification and drought stress results, 12 strains of endophytic fungi with better drought tolerance were selected to study their abilities of dissolving inorganic phosphorus and potassium feldspar powder and producing indole-3-acetic acid (IAA). It was found that the amount of dissolved phosphorus in 7 strains of endophytic fungi was significantly higher than that of CK, and the content of soluble phosphorus was 101.98-414.51 μg. ml-1; 6 endophytic fungi had significantly higher potassium solubilization than CK, and the content of water-soluble potassium ranged from 19.17 to 30.94 mg·l-1; 6 strains have the ability to produce IAA, and the yield of IAA ranged between 0.04 and 0.42 mg. ml-1. This study for the first time identified the existence of endophytic fungi with drought tolerance and growth-promoting function in C. multiflorus, which could provide new direction for plant drought tolerance and growth promotion fungi strain resources. It also provides a theoretical basis for the subsequent application of endophytic fungi of C. multiflorus in agricultural and forestry production to improve plant tolerance.

The potential for endophytes to initiate changes in host secondary metabolism has been well documented. However, the mechanisms underlying endophyte-plant metabolic interactions are still poorly understood. Here, we analysed the effects of fungal endophytes on the metabolite profiles of grape cells from two cultivars: 'Cabernet Sauvignon' (CS) and 'Rose honey' (RH). Our results clearly showed that co-culture with endophytic fungi greatly modified the metabolic profiles in grape cells of both varieties. Treatments with endophytic fungal strains caused the numbers of detected metabolites to vary from 10 to 19 in CS cells and from 8 to 14 in RH cells. In addition, 5 metabolites were detected in all CS cell samples, while 4 metabolites were detected in all RH cell samples. Some endophytic fungal strains could even introduce novel metabolites into the co-cultured grape cells. The metabolic profiles of grape leaves shaped by endophytic fungi exhibited host selectivity and fungal strain specificity. In this assay, the fungal strains RH32 (Alternaria sp.) and MDR36 (Colletotrichum sp.) triggered an increased response of the detected metabolites, including the greatest increase in the metabolite contents in grape cells of both cultivars. No obvious effects in terms of metabolite numbers and contents in grape cells when co-cultured with fungal strains RH7 (Epicoccum sp.) and RH48 (Colletotrichum sp.) were observed. The results of this experiment suggest that endophytic fungi could be used to control the metabolic profiles of grapes and thus increase grape quality.

Ralstonia solanacearum has been known to cause bacterial wilt disease on chili pepper. Despite many reports on the potential use of endophytic fungi to induce plant resistance, its utilization to suppress bacterial wilt disease of chili has not been widely reported. The aims of this research was to screen potential endophytic fungi that may increase chili resistance against bacterial wilt disease. Selection of endophytic fungi was done using in vivo and antibiosis test. Strains of fungi were considered the most potent in suppressing the development of bacterial wilt in chilli were identified. Ten out of 62 isolates of endophytic fungi gave the highest suppression on chilli’s bacterial wilt disease. Most strains of endophytic fungi were able to suppress the development of bacterial wilt disease, but not always positively correlated to the vegetative and generative growth of chilli. Based on the level of disease intensity and the growth of plants were obtained three strains of endophytic fungi that considered potentially suppress the incidence of bacterial wilt disease. The three isolates was identified as Fusarium solani f.sp. phaseoli (AC-2.13 and AC-4.4) and Trichoderma asperellum (AC-3.18) using morphology and molecular characters. Although all three selected isolates were able to suppress bacterial wilt disease in this study, but application of F. solani f.sp. phaseoli should be considered in practical use since it is generally known as the causal agent of root rot disease of beans