Kiyoon Kim

Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation.

Arbuscular Mycorrhizal Fungi Community Structure, Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity.

Psychrotolerant Endophytic Pseudomonas sp. Strains OB155 and OS261 Induced Chilling Resistance in Tomato Plants (Solanum lycopersicum Mill.) by Activation of Their Antioxidant Capacity.

Studies on chilling stress damage and its mitigation through microorganisms in members of family Solanaceae is limited, despite their economic importance. We studied chilling stress alleviation in tomato plants colonized by psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp and P. frederiksbergensis OS261-gfp. Log phase cultures of bacterial strains were coated on surface-sterilized seeds (bacterization) before sowing and nonbacterized (control) seeds were coated with sterile bacterial growth medium. All plants were grown at temperatures of 30 and 25°C and at the end of 4 weeks, chilling treatment (12 and 10°C) was imposed for 1 week on half of the bacterized and control plants. Under normal conditions (30 and 25°C), no significant difference was observed in antioxidant activity, proline accumulation, and expression of cold acclimation genes in tomato leaf tissues of both control and bacterized plants. However, plants exposed to temperatures of 12 and 10°C were found to decrease in robustness and nutrient uptake, accompanied by increased membrane damage. Chilling resistance in bacterized plants was evident from reduced membrane damage and reactive oxygen species levels, improved antioxidant activity in leaf tissues, and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants. Confocal microscopy confirmed effective colonization and intercellular localization of cold-adapted bacterial strains OB155-gfp and OS261-gfp.

Alleviation of salt stress in maize plant by co-inoculation of arbuscular mycorrhizal fungi and Methylobacterium oryzae CBMB20

Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting bacteria (PGPB) can have multiple impacts on phytohormone production and nutrient uptake to improve plant growth. Co-inoculation of AMF and PGPB can mitigate the effects of salinity in plants. This study was conducted to evaluate the efficacy of co-inoculation of AMF (Glomus etunicatum) and Methylobacterium oryzae CBMB20 to alleviate salt stress and improve maize growth. Single and co-inoculation of AMF and M. oryzae CBMB20 significantly increased dry biomass, AMF root colonization, and nutrient accumulation in maize plants under salt stress. The proline content and Na uptake were significantly reduced in co-inoculation of AMF and M. oryzae CBMB20. In addition, co-inoculation of AMF and M. oryzae CBMB20 (soil) showed higher AMF density and AMF colonization compared to single AMF treatment. Among all treatments, co-inoculation of AMF and M. oryzae CBMB20 (soil) was more effective to ameliorate salt stress and improve plant growth. These results demonstrate that co-inoculation of AMF and M. oryzae CBMB20 (soil application) may be an effective alternative to enhance plant growth under salinity stress.

Spore Associated Bacteria (SAB) of Arbuscular Mycorrhizal Fungi (AMF) and Plant Growth Promoting Rhizobacteria (PGPR) Increase Nutrient Uptake and Plant Growth Under Stress Conditions

Microorganisms present in the rhizosphere soil plays a vital role in improving the plant growth and soil fertility. Many kinds of fertilizers including chemical and organic has been approached to improve the productivity. Though some of them showed significant improvement in yield, they failed to maintain the soil properties. Rather they negatively affected soil eventually, the land became unsuitable for agricultural. To overcome these problems, microorganisms have been used as effective alternative. For past few decades, plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) have been used as effective inoculants to enhance the plant growth and productivity. PGPR improves the plant growth and helps the plant to withstand biotic and abiotic stresses. AM fungi are known to colonize roots of plants and they increase the plant nutrient uptake. Spore associated bacteria (SAB) are attached to spore wall or hyphae and known to increase the AMF germination and root colonization but their mechanism of interaction is poorly known. Better understanding the interactions among AMF, SAB and PGPR are necessary to enhance the quality of inoculants as a biofertilizers. In this paper, current knowledge about the interactions between fungi and bacteria are reviewed and discussed about AMF spore associated bacteria.

Exploring the Potential of Bacteria-Assisted Phytoremediation of Arsenic-Contaminated Soils

Arsenic pollution is a serious global concern which affects all life forms. Being a toxic metalloid, the continued search for appropriate technologies for its remediation is needed. Phytoremediation, the use of green plants, is not only a low cost but also an environmentally friendly approach for metal uptake and stabilization. However, its application is limited by slow plant growth which is further aggravated by the phytotoxic effect of the pollutant. Attempts to address these constraints were done by exploiting plant-microbe interactions which offers more advantages for phytoremediation. Several bacterial mechanisms that can increase the efficiency of phytoremediation of As are nitrogen fixation, phosphate solubilization, siderophore production, ACC deaminase activity and growth regulator production. Many have been reported for other metals, but few for arsenic. This mini-review attempts to present what has been done so far in exploring plants and their rhizosphere microbiota and some genetic manipulations to increase the efficiency of arsenic soil phytoremediation.

Cold Stress Tolerance in Psychrotolerant Soil Bacteria and Their Conferred Chilling Resistance in Tomato (Solanum lycopersicum Mill.) under Low Temperatures

The present work aimed to study the culturable diversity of psychrotolerant bacteria persistent in soil under overwintering conditions, evaluate their ability to sustain plant growth and alleviate chilling stress in tomato. Psychrotolerant bacteria were isolated from agricultural field soil samples colleced during winter and then used to study chilling stress alleviation in tomato plants (Solanum lycopersicum cv Mill). Selective isolation after enrichment at 5°C yielded 40 bacterial isolates. Phylogenetic studies indicated their distribution in genera Arthrobacter, Flavimonas, Flavobacterium, Massilia, Pedobacter and Pseudomonas. Strains OS211, OB146, OB155 and OS261 consistently improved germination and plant growth when a chilling stress of 15°C was imposed and therefore were selected for pot experiments. Tomato plants treated with the selected four isolates exhibited significant tolerance to chilling as observed through reduction in membrane damage and activation of antioxidant enzymes along with proline synthesis in the leaves when exposed to chilling temperature conditions (15°C). Psychrotolerant physiology of the isolated bacteria combined with their ability to improve germination, plant growth and induce antioxidant capacity in tomato plants can be employed to protect plants against chilling stress.

Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in psychrotolerant bacteria modulates ethylene metabolism and cold induced genes in tomato under chilling stress.

The role of stress induced ethylene under low temperature stress has been controversial and hitherto remains unclear. In the present study, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) gene, acdS expressing mutant strains were generated from ACCD negative psychrotolerant bacterial strains Flavobacterium sp. OR306 and Pseudomonas frederiksbergensis OS211, isolated from agricultural soil during late winter. After transformation with plasmid pRKACC which contained the acdS gene, both the strains were able to exhibit ACCD activity in vitro. The effect of this ACCD under chilling stress with regards to ethylene was studied in tomato plants inoculated with both acdS expressing and wild type bacteria. On exposing the plants to one week of chilling treatment at 12/10 °C, it was found that stress ethylene, ACC accumulation and ACO activity which are markers of ethylene stress, were significantly reduced in plants inoculated with the acdS gene transformed mutants. In case of plants inoculated with strain OS211-acdS, ethylene emission, ACC accumulation and ACO activity was significantly reduced by 52%, 75.9% and 23.2% respectively compared to uninoculated control plants. Moreover, expression of cold induced LeCBF1 and LeCBF3 genes showed that these genes were significantly induced by the acdS transformed mutants in addition to reduced expression of ethylene-responsive transcription factor 13 (ETF-13) and ACO genes. Induced expression of LeCBF1 and LeCBF3 in plants inoculated with acdS expressing mutants compared to wild type strains show that physiologically evolved stress ethylene and its transcription factors play a role in regulation of cold induced genes as reported earlier in the literature.

Effect of Salinity on Plants and the Role of Arbuscular Mycorrhizal Fungi and Plant Growth-Promoting Rhizobacteria in Alleviation of Salt Stress

Of the world’s 1.5 billion hectares of cultivable land, 77 million hectares (5 %) do not favor good yields due to high salt content and 20 % of the irrigated agricultural land is adversely affected by salinity. Salt-affected soils are increasing steadily in all continents, in particular in arid and semiarid areas which cover more than 7 % of the total land surface on earth. Due to the severe impact of salinity to crop production, salt stress is considered a major limiting factor in crop production. Under saline conditions plant growth is severely reduced, mainly due to osmotic stress. Low water potential in the roots causes water deficit within the plant. Toxic concentrations of Na+ and Cl− ions in plants affect cell membrane functions. Moreover, high amounts of these ions also reduce metabolic activities in plants which lead to growth inhibition and injury of the foliage. Furthermore, low nutrient uptake potential of root due to salinity stress causes nutrient imbalance and oxidative stress in plants which impede proper growth and development. Due to the scope of salinity affected soils and the deleterious effects of salinity on crop production, methods to remediate and/or improve crop production in salinity impacted soils are necessary. Management of soil salinity through land reclamation or improved techniques of irrigation provides only short-term solution and often expensive. Other methods like plant breeding for salt tolerance have been difficult and slow. The nature of salt tolerance traits and problems in developing appropriate testing environment makes it difficult to obtain. Microorganisms present in the rhizosphere soil play a vital role in improving plant growth and soil fertility. Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that form symbiotic or mutualistic associations with roots of about 80 % of plant species. AMF widely exist in salt-affected soils and they infect the plant root cortical cells. AMF form arbuscules inside the cortical cells and utilize them as nutrient exchange sites. These also form vesicles between the cortical cells where all nutrients are stored. Under soil saline conditions, AMF extend their extraradical hyphae into non-rhizosphere soil to uptake more nutrients. Symbioses of halotolerant bacteria with plant roots also help plants withstand salinity stress. Halotolerant bacteria possess plant growth-promoting characters which may improve plant growth and nutrient uptake. The utilization of microorganisms is an economical and environmental friendly approach to alleviate stress in plants cultivated in salt-impacted agricultural fields.

Genetic Diversity and Association Characters of Bacteria Isolated from Arbuscular Mycorrhizal Fungal Spore Walls.

Association between arbuscular mycorrhizal fungi (AMF) and bacteria has long been studied. However, the factors influencing their association in the natural environment is still unknown. This study aimed to isolate bacteria associated with spore walls of AMF and identify their potential characters for association. Spores collected from coastal reclamation land were differentiated based on their morphology and identified by 18S rDNA sequencing as Funneliformis caledonium, Racocetra alborosea and Funneliformis mosseae. Bacteria associated with AMF spore walls were isolated after treating them with disinfection solution at different time intervals. After 0, 10 and 20 min of spore disinfection, 86, 24 and 10 spore associated bacteria (SAB) were isolated, respectively. BOX-PCR fingerprinting analysis showed that diverse bacterial communities were associated to AMF spores. Bacteria belonging to the same genera could associate with different AMF spores. Gram positive bacteria were more closely associated with AMF spores. Isolated SAB were characterized and tested for spore association characters such as chitinase, protease, cellulase enzymes and exopolysaccharide production (EPS). Among the 120 SAB, 113 SAB were able to show one or more characters for association and seven SAB did not show any association characters. The 16S rDNA sequence of SAB revealed that bacteria belonging to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bactereiodes were associated with AMF spore walls.