Salkowski Reagent Research Articles

Comparative study between Salkowski reagent and chromatographic method for auxins quantification from bacterial production.

The Salkowski reagent method is a colorimetric technique used to determine auxin production, specifically as indole-3-acetic acid (IAA). It was developed to determine indoles rapidly; however, it does not follow Beer's law at high concentrations of IAA. Thus, there could be an overestimation of IAA with the Salkowski technique due to the detection of other indole compounds. This study aims to compare the Salkowski colorimetric method versus a chromatographic method to evidence the imprecision or overestimation obtained when auxins, such as indole-acetic acid (IAA), are determined as traits from promoting growth plant bacteria (PGPB), using ten different strains from three different isolation sources. The analysis used the same bacterial culture to compare the Salkowski colorimetric and chromatographic results. Each bacterium was cultivated in the modified TSA without or with tryptophan for 96 h. The same supernatant culture was used in both methods: Salkowski reagent and ultra-performance liquid chromatography coupled with a Mass Spectrometer (LC-MS/MS). The first method indicated 5.4 to 27.4 mg L-1 without tryptophan in ten evaluated strains. When tryptophan was used as an inductor of auxin production, an increase was observed with an interval from 4.4 to 160 mg L-1. The principal auxin produced by all strains was IAA from that evaluated by the LC-MS/MS method, with significantly higher concentration with tryptophan addition than without. Strains belonging to the Kocuria genus were highlighted by high IAA production. The indole-3-propionic acid (IPA) was detected in all the bacterial cultures without tryptophan and only in K. turfanensis As05 with tryptophan, while it was not detected in other strains. In addition, indole-3-butyric acid (IBA) was detected at trace levels (13-16 µg L-1). The Salkowski reagent overestimates the IAA concentration with an interval of 41-1042 folds without tryptophan and 7-16330 folds with tryptophan as inductor. In future works, it will be necessary to determine IAA or other auxins using more suitable sensitive techniques and methodologies.

Plant Growth Promotion and Biological Control against Rhizoctonia solani in Thai Local Rice Variety "Chor Khing" Using Trichoderma breve Z2-03.

Several strains of Trichoderma are applied in the field to control plant diseases due to their capacity to suppress fungal pathogens and control plant diseases. Some Trichoderma strains also are able to promote plant growth through the production of indole-3-acetic acid (IAA). In southern Thailand, the local rice variety "Chor Khing" is mainly cultivated in the Songkhla province; it is characterized by slow growth and is susceptible to sheath blight caused by Rhizoctonia solani. Therefore, this research aimed to screen Trichoderma species with the ability to promote plant growth in this rice variety and enact biological control against R. solani. A total of 21 Trichoderma isolates were screened for indole compound production using the Salkowski reagent. The Z2-03 isolate reacted positively to the Salkowski reagent, indicating the production of the indole compound. High-performance liquid chromatography (HPCL) confirmed that Z2-03 produced IAA at 35.58 ± 7.60 μg/mL. The cell-free culture filtrate of the potato dextrose broth (CF) of Z2-03 induced rice germination in rice seeds, yielding root and shoot lengths in cell-free CF-treated rice that were significantly higher than those of the control (distilled water and culture broth alone). Furthermore, inoculation with Trichoderma conidia promoted rice growth and induced a defense response against R. solani during the seedling stage. Trichoderma Z2-03 displayed an antifungal capacity against R. solani, achieving 74.17% inhibition (as measured through dual culture assay) and the production of siderophores on the CAS medium. The pot experiment revealed that inoculation with the Trichoderma sp. Z2-03 conidial suspension increased the number of tillers and the plant height in the "Chor Khing" rice variety, and suppressed the percentage of disease incidence (PDI). The Trichoderma isolate Z2-03 was identified, based on the morphology and molecular properties of ITS, translation elongation factor 1-alpha (tef1-α), and RNA polymerase 2 (rpb2), as Trichoderma breve Z2-03. Our results reveal the ability of T. breve Z2-03 to act as a plant growth promoter, enhancing growth and development in the "Chor Khing" rice variety, as well as a biological control agent through its competition and defense induction mechanism in this rice variety.

Influence of IAA-Producing Bacteria and Phosphate Solubilizers from The Rhizosphere of Serbania grandiflora (L.) Pers on The in vitro Germination of Vigna radiata (L.)

This study aims to determine the effect of IAA-producing bacteria and phosphate solvents from the rhizosphere of Serbania grandiflora (L.) Pers on the germination of Vigna radiata (L.). This research began by isolating the rhizosphere bacterium Serbania grandiflora (L.) Pers, which was then characterized by cell morphology, grammatical and morphological characteristics. The ability of bacteria to dissolve phosphate was evaluated qualitatively by observing the presence of clear zones in Pikovskaya media, and quantitatively with a spectrophotometer. The isolate's ability to produce IAA was tested quantitatively using Salkowski reagent, and quantitatively using a spectrophotometer. For the effect of isolates on the growth of green beans, a completely randomized design (CRD) research design was used, with 4 treatments, namely P0 (sterile distilled water); P1 (IAA-producing bacterial culture); P2 (phosphate solubilizing bacterial culture); P3 (IAA producer and phosphate solubilizer). Vigna radiata L. seedling growth test was carried out in vitro on Murphy's medium. The research data were analyzed using the one-way ANOVA test. The results showed that 5 isolates had the ability to dissolve phosphate, with a phosphate solubility index ranging from 1.70-2.14, where isolate RT7 showed the highest value. The isolate's ability to produce IAA at concentrations ranged from 7.1 - 62.7 ppm, with the highest value shown by isolate RT6. The results showed that treatment with IAA-producing bacterial isolates plus phosphate solubilization properties showed a significantly different effect on Vigna radiata L. plant height compared to other treatments.

Colletotrichum truncatum, an endophytic fungus derived from Musa acuminata (AAA group): antifungal activity against Aspergillus isolated from COVID-19 patients and indole-3-acetic acid (IAA) production

Fungal endophyte is a fungal that lives in plant organism as mutualism association. The role of fungal endophyte is a growth promoter or/and microbial pathogen inhibitor. This study investigated antifungal activity of Colletotrichum truncatum E10, an endophytic fungus derived from Musa acuminata (AAA group), against 7 isolates of Aspergillus obtained from lower respiratory samples of COVID-19 patients. In addition, IAA production of this strain was also observed. All isolates of Aspergillus were identified using MALDI-TOF MS. The fungal endophyte, C. truncatum E10, was screened for IAA induction with and without 0.1, 2 and 8 mg/mL of L-tryptophan based on colorimetric method using Salkowski reagent which produced pinkish to reddish solution indicating the presence of IAA. Antagonist activity was based on dual culture assay measured in colony growth inhibition (CGI). C. truncatum E10 produced the highest IAA concentration of 112.81±0.12 µg/mL when 8 mg/mL of L-tryptophan added. The strong antagonist activities were shown by C. truncatum E10 against 5 Aspergillus isolates including 2 A. fumigatus: sp442/6 (CGI=57.83±5.11%) and sp269/11 (CGI=53.01±8.52%), 1 A. niger sp26/7 (CGI=57.83±15.33%) and 2 A. flavus: sp26/7 (CGI=56.63±13.63%) and sp36/7 (CGI=57.23±0.85%), whereas the colony growth inhibition (CGI) of other 2 isolates including A. fumigatus sp567/6 and A. flavus sp269/11 were less than 50%. In this study, C. truncatum E10 produced substances that inhibited human fungal pathogen including A. fumigatus, A. flavus and A. niger. Moreover, it can produce IAA activity. Further investigations are being conducted to expand the plant growth promotion effects and determine IAA biosynthesis pathway. For antifungal activity, the bioactive metabolites produced by this endophytic fungal isolate should be characterized to specify the effective compounds. Asian Australas. J. Biosci. Biotechnol. 2023, 8 (2), 23-29

Whole-Genome Sequence Insight into the Plant-Growth-Promoting Bacterium Priestia filamentosa Strain AZC66 Obtained from Zygophyllum coccineum Rhizosphere.

This study aimed to isolate, screen the plant-growth-enhancing features, and explore the whole-genome sequence of AZC66 isolated from the rhizosphere of Zygophyllum coccineum and determine its biostimulating effects on the growth of cowpea under greenhouse conditions. Salkowski reagent was used to measure AZC66's indole acetic acid production. AZC66's inorganic phosphate solubility on Pikovskaya agar was evaluated using tricalcium phosphate. The results indicated the ability of AZC66 to fix nitrogen, produce IAA (66.33 ± 0.44 μg mL-1), solubilize inorganic phosphate, and exhibit the activity of ACC deaminase (278.40 ± 21 mol -ketobutyrate mg-1 h-1). Cowpea's root and shoot dry weights were also significantly increased after in vitro inoculation with AZC66. The identity of AZC66 was confirmed as Priestia filamentosa, and 4840 genes were predicted in its genome. The gene sequences were compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the results showed that the top three pathways wherein the maximum number of genes are involved are signaling and cellular processes, genetic information processing, and carbohydrate metabolism. The genome sequencing of the strain AZC66 revealed a number of genes implicated in plant biostimulation activities such as nitrogen fixation (nifU), phytohormone synthesis (trpAB genes), phosphate solubilization (PhbCEF, pstABCS, and phoU), and siderophore formation (FbpA, feoAB, and fetB). The AZC66 genome contained numerous genes involved in nitrogen metabolism, nitrogen regulation, and the nitrate reduction pathway. The phenazine biosynthetic gene in AZC66 demonstrated biocontrol and soil survival properties. The trehalose synthesis genes in AZC66 may help plants resist osmotic and salt stress. The discovery of glycine betaine, cold shock, and heat shock protein genes demonstrated that AZC66 could withstand harsh conditions. AZC66 might be used to create robust, sustainable biological fertilizers for future agricultural use in Saudi Arabia. Furthermore, the predicted adaptable metabolic pathways might serve as the basis for potential biotechnological applications in agriculture and industry.

Microassay validation for bacterial IAA estimation as a new fine-tuned PGPR screening assay

The detection and quantification of Indole -3 Acetic Acid (IAA) produced by Plant growth promoting rhizobacteria (PGPR) rely on a standard well-documented assay, which remains time-consuming, laborious, and costly. These drawbacks led to sway interest to economic and reliable assays. The aim of this work is to validate and standardize a fast, reliable, and cost-effective microassay to quantify IAA produced by bacteria with an easy microplate method. In order to validate the accuracy of the IAA microplate assay, bacterial samples from different genera were assayed using two methods: the conventional IAA estimation assay and the IAA micro- assay. The microassay shows a prominent reduction in used bacterial supernatant volume as well as Salkowski reagent volume of about 92.5%. It is considerably cheaper than the conventional one of around 56%. The newly performed microplate assay is 23 times faster. The result of IAA quantitative analysis for 13 bacterial strains showed that Bacillus muralis and Bacillus toyonensis produced the highest IAA concentration (23.64±0.003μg/ml and 23.35±0.006μg/ml, respectively). The obtained data from both methods were highly correlated with an R-value of 0.979. The microassay offers the ability to read the optical density of all samples simultaneously since used volumes of bacterial supernatants and Salkowski reagent were minimized to place the mixture in 96-well microplates, which reduces greatly required labor. Furthermore, the application of the IAA micro-plate assay reduces drastically the reagent waste and toxicity hazard of Salkowski reagent in the environment, thus, we can classify it as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA). The IAA microassay is a, reliable, rapid and cost-effective and eco-friendly method to screen plant growth promoting potential of more than 23 bacterial strains by microplate. It could be an alternative for the conventional IAA assay as a routine research tool.

Increasing Plant Health using Plant Growth Regulator from Rice Rhizobacteria

Plant health is a very important factor in the agriculture system. The presence of plant hormones such as Auxin, gibberellic acid, and siderophores with variation numbers in the plant gives different effects on plant health. Related to the environment rich with rhizobacteria, they have a unique function because rhizobacteria can produce a chemical compound known as Plant Growth Promoting Rhizobacteria (PGPR). The purpose of the research is to study the production of auxin, gibberellic acid, and siderophore from rice rhizobacteria as the source of potential hormones for plant growth. Ten rhizobacteria isolates have been isolated from the rice rhizosphere. They are potential candidates as biofertilizers and biopesticide agents. The hormone production from each isolate was tested by spectrophotometric methods: Auxin at a wavelength of 535 nm with Salkowski's reagent, gibberellic acid at 254 nm, and siderophores at 560 nm with Hardaway's reagent. The results showed the highest auxin hormone was obtained from isolate 10 (3.374 ppm), the highest gibberellic acid from isolate 4 (3.960 ppm), and the highest siderophores hormone from isolate 2 (2.910 ppm). The conclusion of the research is, plant growth regulator from rice rhizobacteria has the potential increasing plant health.

Перспективы использования микробных препаратов для снижения окислительного стресса сельскохозяйственных растений

Anthropogenic factors expose agricultural plants to abiotic and biotic stresses, one of which is oxidative stress. Oxidative stress changes cell metabolism, as well as inhibits plant growth and development. Microbial treatment is an environmentally safe method of oxidative stress prevention. The research objective was to study the antioxidant activity of microflora native to coal dumps in order to combat the oxidative stress in crops.
 The study featured microorganisms isolated from technogenically disturbed soils. Pure bacterial cultures were isolated by deep inoculation on beef-extract agar. A set of experiments made it possible to define the cultural, morphological, and biochemical properties of cell walls. The antioxidant activity and the amount of indole-3-acetic acid were determined on a spectrophotometer using the ABTS reagent and the Salkowski reagent, respectively. The isolated microorganisms were identified on a Vitek 2 Compact device. The biocompatibility of strains was tested by dripping, while the increase in biomass was measured using a spectrophotometer.
 The study revealed ten microbial strains with antioxidant activity ranging from 67.21 ± 3.08 to 91.05 ± 4.17%. The amount of indole-3-acetic acid varied from 8.91 ± 0.32 to 15.24 ± 0.69 mg/mL. The list of microorganisms included Klebsiella oxytoca, Enterobacter aerogenes, Pseudomonas putida, and Bacillus megaterium. The consortium of P. putida and E. aerogenes demonstrated the best results in antioxidant activity, indole-3-acetic acid, and biomass. Its ratio was 2:1 (94.53 ± 4.28%; 15.23 ± 0.56 mg/mL), while the optical density was 0.51 ± 0.02. Extra 2% glycine increased the antioxidant activity by 2.34%, compared to the control. Extra 0.5% L-tryptophan increased the amount of indole-3-acetic acid by 3.12 mg/mL and the antioxidant activity by 2.88%.
 The research proved the antioxidant activity of strains isolated from microflora native to coal dumps. The consortium of P. putida and E. aerogenes (2:1) demonstrated the best results. Further research will define its ability to reduce oxidative stress in plants.

Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings

BackgroundEarly blight (EB), caused by Alternaria solani, is one of the alarming diseases that restrict tomato production globally. Existing cultural practices and fungicide applications are not enough to control early blight diseases. Therefore, the study aimed to isolate, identify, and characterize an endophytic Streptomyces exhibiting the potential to control early blight in tomato and also promote plant growth.ResultsFrom a Citrus jambhiri leaf, an endophytic Streptomyces sp. with antagonistic activity against Alternaria solani, Colletotrichum acutatum, Cladosporium herbarum, Alternaria brassicicola, Alternaria sp., Fusarium oxysporum and Fusarium sp. was isolated. It was identified as a Streptomyces sp. through 16S ribosomal DNA sequence analysis and designated as SP5. It also produced indole acetic acid which was confirmed by Salkowski reagent assay, TLC and HPLC analysis. Treatment of pathogen infected plants with Streptomyces sp. SP5 antagonists (culture cells/culture supernatant/solvent extract/ acetone precipitates) decreased the early blight disease incidence and significantly increased the various agronomic traits.ConclusionThe present study concluded that Streptomyces sp. SP5 possessed antifungal activity against different fungal phytopathogens and had significant potential to control early blight disease and promote plant growth.

Production and Characterization of Indole Acetic Acid of Endophytic Bacteria isolated from Kalanchoe pinnata (Lam.) in Optimized Media

Objectives: To optimize, quantify and characterize indole acetic acid (IAA) production from endophtic bacteria isolated from the leaves of Kalanchoe pinnata (Lam.). Methods: The selected endophytic bacteria are identified by biochemical, morphological and 16SrRNA sequencing analysis. Endophytes are screened for IAA using Salkowski reagent and media optimization is carried out for maximum IAA yield and quantification was done with UV Spectrophotometer at 30 and 120 minutes. The mean  standard error of each sample for three triplicates is calculated from mean value of the samples followed by normal distribution. The characterization of IAA was studied with ethyl acetate extract of endophytes using TLC and HPLC. Findings: The selected endophytes are identified as Bacillus thuringiensis, Bacillus paranthracis, Staphylococcus xylosus and Bacillus cereus. Optimized yeast malt dextrose (YMD) broth containing mannitol, ammonium nitrate, and 0.2% of L-tryptophan at pH 8 and 30◦ C temperature for 72 h was used to obtain maximum IAA production from these endophytes. B. paranthracis produced 82.47330.0728mg/ml and 83.56670.457mg/ml, S. xylosus produced 93.120.261mg/ml and 94.76670.285mg/ml, B. cereus produced 24.4 0.113mg/ml and 25.0667 0.581mg/ml and B. thrungiensis produced 4.13330.131mg/ml and 50.226mg/ml of IAA at 30 and 120 minutes, respectively. B. paranthracis and S. xylosus were producing potential IAA with Rf 0.91 and 0.95 respectively compared with standard Rf 0.95. The HPLC analysis of Bacillus paranthracis and Staphylococcus xylosus were confirmed for the presence of IAA with the retention time of 5.053 and 5.58 minutes respectively compared to the retention time of the standard. Novelty: Endophytic bacteria notable in the leaves of Kalanchoe pinnata (Lam.) produce indole acetic acid in measurable quantity by UV Spectrometry and HPLC analysis, that can be replace for unstable and expensive synthetic IAA using costly chemicals. This is best pilot scale production of IAA using endophytes in the field of white biotechnology. Keywords: Endophytes; Kalanchoe pinnata; Indole Acetic Acid; TLC; HPLC

In Vitro Antioxidant, Cytotoxic, Thrombolytic Activities and Phytochemical Evaluation of Methanol Extract of the Ampelocissus Barbata (Wall.) Leaves

Context: Oxidative stress and pertaining counterbalance mechanism are actively working in the living organisms. The overproduction of reactive oxygen species (ROS) in the ongoing equipoising process requires to be compensated by strong antioxidants. Plants as a rich source of antioxidants not only reduce oxidative stress but also possess cytotoxic, thrombolytic and phytochemical potentials. Aims: To find out the antioxidant, cytotoxic, thrombolytic and phytochemical capabilities of the methanolic extracts of Ampelocissus barbata (Wall.) leaves. Methods and Material: Assessment of the in vitro antioxidant activity of extract was carried out using DPPH radical scavenging assay, determination of reducing power capacity and total phenolic content. The thrombolytic activity was assessed by disintegration of clot and prospective phytochemical activities were by standard qualitative analysis such as Mayer’s, Dragendroff’s Wagner’s and Hager’s Reagent test for alkaloids; Libermann-Burchared and Salkowski Reagent tests for steroid and terpenoids; Molish Reagent, Benedict’s Reagent, Fehling’s Solution A & B reagent test for carbohydrates; Ferric Chloride (5%) Solution, Potassium Dichromate (10%) Solution tests for tannins; Shinoda test and Alkaline reagent test for Flavonoids; Froth tests & Haemolysis test for Saponins. Statistical analysis used: The statistical analysis was carried out using GraphPad Prism and Microsoft excel Results: Appreciable DPPH radical scavenging activity of the extract was observed with the IC50 value of 107.47±1.46 µg/ml. A significant correlation was found between the standard ascorbic acid (AA) and the plant extracts at the p˂0.05 for the reducing power assay where, the activity increased with the concentration of the extracts and the highest absorbance value was 3.025±0.15 and 1.826±0.006 for the AA and the extracts respectively. The plant also accommodates a considerable amount of polyphenols, reflected in the value of gallic acid equivalent 277.397±0.419 mg/ml. Finally, the percentage (%) of clot lysis for the thrombolytic activity was revealed to be 7.031±0.697, 35.297±1.307, and 75.083±0.599 for the water (negative control), extract, and the standard Streptokinase respectively. The study revealed the presence of phytochemicals namely alkaloids, flavonoids, tannins and glycosides. Conclusions: The study disclosed the promising in vitro activity of the plant, which necessitates the further analysis for the isolation and evaluation of the active principles.

Environmental Factors Modulating Indole-3-Acetic Acid Biosynthesis by Four Nitrogen Fixing Bacteria in a Liquid Culture Medium

This study evaluated the effects of some environmental conditions on IAA biosynthesizing capacity of four nitrogen fixing bacteria, namely Paenibacillus cineris TP-1.4, Bacillus megaterium MQ-2.5, Klebsiella pneumoniae OM-17.2, and Pseudomonas boreopolis CP-18.2. Carbon source, pH, NaCl, and tryptophan supplement treatments were set to investigate the effects of those environmental factors on IAA synthesis. The IAA synthesizing capacity of bacterial strains in liquid medium was measured spectroscopically following incubation by Salkowski's reagent method. The results showed that, under the sucrose amendment, the IAA concentrations produced by all four bacterial strains were significantly higher than those of the other four carbon source added treatments. Two of the four bacterial strains produced the highest yield of IAA in liquid medium at pH 7 (TP-1.4 and OM-17.2), whereas pH 8 was optimum for the other two strains (MQ-2.5 and CP-18.2). The MQ-2.5 strain could synthesize IAA fairly well in up to 5% NaCl and produced the highest amount of IAA with 1% NaCl. Furthermore, IAA synthesizing capability of tested bacterial strains increased sharply along with increasing tryptophan content in culture medium except for the TP-1.4 strain. From the current study, these isolates emerged as possible alternatives for future IAA production for plant growth and yield enhancement. Hence, they have a great potential to be used as bio-inoculants for plant growth promotion in eco-friendly and sustainable agriculture.

Quantitative Estimation of Indole-3-acetic Acid in Shoot Tips and Young Leaves of Tecoma stans and Ixora coccinea from Polluted and Control Regions

Plants are sensitive to pollution and especially air pollution can alter the physiological processes of plants affecting growth patterns. Study was carried out to determine the IAA production and response of plants exposed to vehicular emission. Two plant species namely Tecoma stans and Ixora coccinea which were exposed to roadside vehicular pollution were taken and control plants were collected from the college campus (unpolluted zone) and Estimation of IAA is carried out using Salkowski reagent, which upon reaction with IAA, yields pink color, due to AA complex formation and reduction of Fe+3. A decrease and increase in IAA concentration was observed in Tecoma stans and Ixora cocenia respectively growing in pollution zones as compared to control zones clearly demonstration air pollution stress.

ISOLATION AND CHARACTERIZATION OF IAA-PRODUCING RIZOSPHERE BACTERIA FROM RICE PLANTS (ORYZA SATIVA L.) IN KEDUNGPANI URBAN VILLAGE, SEMARANG CITY

Rhizosphere bacteria live in the soil around plant roots. Various rhizosphere bacteria are able to produce the hormone Indole-3-Acetic Acid (IAA) which stimulates plant growth. This study aims to isolate and characterize IAA-producing rhizosphere bacteria in rice plants in the rice fields of Kedungpani urban village, Semarang city. Bacterial enumeration was carried out using the total plate count (TPC) method. The isolates obtained were characterized by observation of colony and cell morphology, gram staining, endospore staining, catalase test, and sugar fermentation test. The ability to produce the IAA hormone was tested using Salkowski's reagent. The enumeration results showed that the total bacteria in the sample was 2.9 x 105 CFU / g. A total of 10 isolates were obtained and were able to produce the IAA hormone with various concentrations. Isolates KP1, KP2, KP3, KP4, KP5, KP6, KP9, KP13, KP14, and KP15 produced IAA with concentrations of 3.389, 5.111, 3.000, 1.667, 1.944, 5.056, 5.444, 4.500, 5.056, and 10.833 ppm, respectively. The results showed that there were 5 isolates with the highest IAA concentrations, namely KP2, KP6, KP9, KP14, and KP15 which had the potential to be used as biostimulant agents for plants.

Selection of fungi that potentially produces IAA (Indole Acetic Acid) hormone origin of Takalar sugar factory waste

This research aimed to obtain fungi isolates from the laboratory collection Mycology Department of Plant Pests and Diseases Hasanuddin University which has the potential to produce hormones Indole Acetic Acid (IAA). The isolate used is the result of isolation from Takalar sugar factory waste. Measurement The fungi isolate was inoculated in the media of Potato Dextrose Broth (PDB) enriched L-Tryptophan and then added the Salkowski Reagent. The results showed a change in pink color showed the fungi can produce the IAA hormone. High and low content of IAA produced by fungi isolates was shown with pink color. The pinker the suspense color, the higher the IAA content. IAA concentration produced by the isolates was measured using a spectrophotometer with a wavelength of 520 nm. The IAA value is calculated by comparing it with the absorbance value of the standard curve. The results showed that the isolate D4 code produced the highest IAA hormone concentration of 10.17 ppm.

Bacterial Endophytes of Spring Wheat Grains and the Potential to Acquire Fe, Cu, and Zn under Their Low Soil Bioavailability.

Simple SummaryUnmasking the overall endophytic bacteria communities from wheat grains may help to identify and describe the microbial colonization of bread and emmer varieties, their link to the bioactive compounds produced, and their possible role in mineral nutrition. The possibility of using microorganisms to improve the microelemental composition of grain is an important food security concern, as approximately one-third of the human population experiences latent starvation caused by Fe (anemia), Zn, or Cu deficiency. Four wheat varieties from T. aestivum L. and T. turgidum subsp. dicoccum were grown in field conditions with low bioavailability of microelements in the soil. Varietal differences in the yield, yield characteristics, and the grain micronutrient concentrations were compared with the endophytic bacteria isolated from the grains. Twelve different bacterial isolates were obtained that represented the genera Staphylococcus, Pantoea, Sphingobium, Bacillus, Kosakonia, and Micrococcus. All studied strains were able to synthesize indole-related compounds (IRCs) with phytohormonal activity. IRCs produced by the bacterial genera Pantoea spp. and Bacillus spp. isolated from high-yielding Oksamyt myronivs’kyi and Holikovs’ka grains may be considered as one of the determinants of the yield of wheat and its nutritional characteristics.Wheat grains are usually low in essential micronutrients. In resolving the problem of grain micronutritional quality, microbe-based technologies, including bacterial endophytes, seem to be promising. Thus, we aimed to (1) isolate and identify grain endophytic bacteria from selected spring wheat varieties (bread Oksamyt myronivs’kyi, Struna myronivs’ka, Dubravka, and emmer Holikovs’ka), which were all grown in field conditions with low bioavailability of microelements, and (2) evaluate the relationship between endophytes’ abilities to synthesize auxins and the concentration of Fe, Zn, and Cu in grains. The calculated biological accumulation factor (BAF) allowed for comparing the varietal ability to uptake and transport micronutrients to the grains. For the first time, bacterial endophytes were isolated from grains of emmer wheat T. turgidum subsp. dicoccum. Generally, the 12 different isolates identified in the four varieties belonged to the genera Staphylococcus, Pantoea, Sphingobium, Bacillus, Kosakonia, and Micrococcus (NCBI accession numbers: MT302194—MT302204, MT312840). All the studied strains were able to synthesize the indole-related compounds (IRCs; max: 16.57 µg∙mL−1) detected using the Salkowski reagent. The IRCs produced by the bacterial genera Pantoea spp. and Bacillus spp. isolated from high-yielding Oksamyt myronivs’kyi and Holikovs’ka grains may be considered as one of the determinants of the yield of wheat and its nutritional characteristics.

The measurement of indole acetic acid from rhizosphere bacteria

In Indonesia, synthetic chemical fertilizers are generally used to spur plant growth. The use of synthetic chemical fertilizers is known to reduce soil quality, causing resistance to pests and plant diseases. One of the natural potencies that can be used as growth promoters is Indole Acetic Acid (IAA) producing bacteria. The objective of this study was to measure the levels of Indole Acetic Acid (IAA) from the rhizosphere bacterial isolate of green beans which is thought to be able to produce Indole Acetic Acid (IAA). The method used was a descriptive approach, including the isolation of green bean rhizosphere bacteria, measurement of Indole Acetic Acid (IAA) levels in bacterial isolate, and data analysis. Green bean rhizosphere bacterial isolate produced Indole Acetic Acid (IAA) with an indication of a color change after the addition of the Salkowski reagent, and quantitatively the results of calculations in the standard curve equation for Indole Acetic Acid (IAA) obtained a value of Indole Acetic Acid (IAA) levels was 50.91 ppm.Keywords: Indole acetic acid, rhizosphere, bacteria

Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity

Endophytes are growth-promoting agents capable of synthesizing phytohormones, uptaking nutrients, and controlling pathogens. There is a strong potential to exploit them in the agriculture field like biofertilizers and biocontrol agents. In this work, we aimed to evaluate endophytic fungi isolated from Pachystachys lutea for their potential to solubilize phosphate, synthesise indole acetic acid (IAA), antagonize phytopathogens, and promote plant growth under greenhouse conditions. The phosphate solubilization efficiency was assessed on Pikovskaya’s agar medium. For analysis of IAA production, mycelia plugs of endophytes were cultured in Potato Dextrose Broth medium supplemented with L-tryptophan, with Salkowski Reagent, and the absorbance of the culture was measured. The antagonism evaluation of strain Alternaria sp. PL75 against phytopathogens was performed using the paired-culture technique. The promotion of plant growth provided by Alternaria sp. PL75 was evaluated in tomato plants. All strains evaluated were able to solubilize phosphate; however, the strain Alternaria sp. PL75 was the most effective (4.29). Two strains, Nemania sp. PL27 and Alternaria sp. PL75, produced 1.86 and 1.73 μg mL-1 of IAA, respectively. In the antagonism assay, the endophyte Alternaria sp. PL75 and its fungal extract showed the best results against the pathogen Moniliophthora perniciosa. The greenhouse experiment result showed the endophyte Alternaria sp. PL75 increased the plantlets emergency speed index and the percentage of germination from 60 to 81.63%. It was also observed a statistical significance in the shoot length of the treated plants with the endophyte suspension (55.38 cm) compared to the control (41.67 cm).

The Relationship between Rhizobacteria Auxin Production and Wheat Biomass Productivity is Negative

ABSTRACT Auxin, a plant growth-promoting hormone, is known to be produced by many if not all bacteria. Research has shown that bacteria produced auxin is associated with both positive and negative plant growth effects. Here we determined the relationship between auxin production capacity and wheat biomass productivity in culturally isolated bacteria. A total of 4320 individual bacteria were isolated and characterized based on colony size as either fast (>2 mm) or slow (<2 mm) growing, from the rhizospheres and endorhizospheres of wheat plants grown in two soils that differ in pH and texture. The bacteria were assayed for auxin production based on a Salkowski reagent microplate assay. The relationship between auxin production and wheat biomass was largely negative under all conditions and characterizations, and non-significant (R2 0.04 to 0.26). When examined across all treatments, the lowest auxin producers originated from the highest biomass plants and the highest auxin producers from the lowest biomass plants (p value < .0001). Fast growing colonies produced significantly higher auxin production than slow growing colonies, overall (p value ≤ 0.005). The results suggest that auxin production by culturally isolated bacteria, as determined by the Salkowski reagent, is negatively associated with wheat biomass productivity.

ISOLASI DAN KARAKTERISASI KEMAMPUAN BAKTERI ENDOFIT SORGUM MANIS FS501 SEBAGAI PENDUKUNG PERTUMBUHAN TANAMAN

This study aims to isolate endophytic bacteria of sweet sorghum plants and characterize their ability as plant growth promoting. The characters tested in this study were the ability of endophytic bacteria in N fixation, phosphate dissolving, and IAA production to be developed as biological fertilizer agents. Twenty-four isolates were isolated from the roots, stems and shoots of sweet sorghum. The results of bacterial DNA fingerprint screening showed that 11 groups of endophytic bacteria had different fingerprints. Isolates capable of N fixation were grown on LGI media and showed a change in the color of the medium. The nifH gene detection is also carried out to determine endophytic diazotrophic bacteria. Isolate bacteria that can dissolve inorganic phosphate were tested using Pikovskaya media. Testing the ability of isolates to produce IAA was carried out by adding Salkowski's reagent to the bacterial culture and measured quantitatively with a λ 530 nm spectrophotometer. The results showed that two endophytic bacterial isolates proved to be diazotrophic and three isolates were able to dissolve phosphate, while one isolate was able to produce IAA. PA2 isolate showed ability in all the characters tested, namely N fixation, phosphate solvent and IAA producer.