Response of endophytic bacterial communities in banana tissue culture plantlets to Fusarium wilt pathogen infection

Endophytic bacteria reside within plant hosts without having pathogenic effects, and various endophytes have been found to functionally benefit plant disease suppressive ability. In this study, the influence of banana plant stress on the endophytic bacterial communities, which was achieved by infection with the wilt pathogen Fusarium oxysporum f. sp. cubense, was examined by cultivation-independent denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA directly amplified from plant tissue DNA. Community analysis clearly demonstrated increased bacterial diversity in pathogen-infected plantlets compared to that in control plantlets. By sequencing, bands most similar to species of Bacillus and Pseudomonas showed high density in the pathogen-treated pattern. In vitro screening of the isolates for antagonistic activity against Fusarium wilt pathogen acquired three strains of endophytic bacteria which were found to match those species that obviously increased in the pathogen infection process; moreover, the most inhibitive strain could also interiorly colonize plantlets and perform antagonism. The evidence obtained from this work showed that antagonistic endophytic bacteria could be induced by the appearance of a host fungal pathogen and further be an ideal biological control agent to use in banana Fusarium wilt disease protection.     

Diversity and chemotaxis of soil bacteria with antifungal activity against Fusarium wilt of banana

The chemotactic response of bacteria to root exudates plays an important role in the colonization of bacteria in the rhizosphere. In this study, 420 strains of antifungal bacteria against Fusarium oxysporum f. sp. cubense (Foc) were screened for chemotaxis based on a cheA molecular diagnostic method. A total of 124 strains with antifungal efficiencies of 27.26-67.14?% generated a characteristic band of cheA. The chemotaxis of 97 bacterial strains producing a cheA band was confirmed using the drop assay and swarm plate assay using catechol, p-hydroxybenzoic acid, salicylic acid, and asparagine as the attractants. A phylogenetic analysis based on restriction fragment length polymorphisms (RFLPs) and 16S rDNA sequences indicated that the 124 chemotactic antagonists of Foc were affiliated with 18 species of Paenibacillaceae, Bacillaceae, Streptomycineae, Enterobacteriaceae, and Pseudomonadaceae. The chemical composition of banana root exudates were analyzed by GC-MS, and 62 compounds, including alkanes, alkenes, naphthalenes, benzenes, and alcohols, were evaluated. Five representative antagonists of Foc showed 1.76- to 7.75-fold higher chemotactic responses than the control to seven compounds in banana root exudates, as determination by capillary assays.     

An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing N ⁶-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.     

Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth

A total of 40 endophytic bacterial isolates obtained from banana tree roots were characterized for their biotechnological potential for promoting banana tree growth. All isolates had at least one positive feature. Twenty isolates were likely diazotrophs and formed pellicles in nitrogen-free culture medium, and 67% of these isolates belonged to the genus Bacillus sp. The isolates EB-04, EB-169, EB-64, and EB-144 had N fixation abilities as measured by the Kjeldahl method and by an acetylene reduction activity assay. Among the 40 isolates, 37.5% were capable of solubilizing inorganic phosphate and the isolates EB-47 and EB-64 showed the highest solubilization capacity. The isolate EB-53 (Lysinibacillus sp.) had a high solubilization index, whereas 73% of the isolates had low solubilization indices. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in 40% of the isolates. The isolate EB-40 (Bacillus sp.) produced the highest amount of IAA (47.88 μg/ml) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The isolates EB-126 (Bacillus subtilis) and EB-47 (Bacillus sp.) were able to simultaneously fix nitrogen, solubilize phosphate and produce IAA in vitro. The results of this study demonstrated that the isolates analyzed here had diverse abilities and all have the potential to be used as growth-promoting microbial inoculants for banana trees.     

NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation

NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1(+) NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1(-) NK cells are precursors of KLRG1(+) NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1(+) NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45(-/-) mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.     

Effect of the combination of bio-organic fertiliser with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease,crop production and banana rhizosphere culturable microflora

Soil amended with organic amendments has been suggested to be a strategy for managing the Fusarium wilt disease which severely hindered the banana production. The effects of four fertilisation regimes, including chemical fertiliser, manure composts and bio-organic fertiliser (BIO) containing Bacillus amyloliquefaciens NJN-6 for 2-year continuous application on the banana Fusarium wilt disease incidence, crop yield and rhizosphere culturable microbial community were investigated. To explore the soil microflora, plate counting method, in vitro screening method for antagonism, eco-physiological index and culture-dependent denaturing gradient gel electrophoresis method (CD DGGE) were used. The highest banana yield, culturable bacteria, actinobacteria and Bacillus populations, culturable bacteria to fungi (B/F) value, antagonistic Bacillus ratio and lowest Fusarium wilt disease incidence were observed in the BIO treatment. Based on CD DGGE results, the BIO application significantly altered the soil bacteria structure and showed highest richness and diversity. The phylogenetic analysis of the selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas, Chitinophaga, the species Bacillus flexus and uncultured Bacillus. All the results showed that 2-year continuous application of BIO containing B. amyloliquefaciens NJN-6 more effectively controlled Fusarium wilt disease and improved fruit yields under field conditions and modulated banana rhizosphere microflora.     

Isolation and characterization of fluorescent Pseudomonas associated with the roots of rice and banana grown in Sri Lanka

Bacterial populations in different parts of the rhizosphere of rice and banana in Sri lanka were examined. On rice, the number of aerobic bacteria and the population of fluorescent bacteria were higher in the rhizoplane as compared to the exorhizosphere. However, the opposite was observed with banana. Percentage of fluorescent bacteria was significantly higher on banana (10.8%) than on rice from the wet and dry zones of Sri Lanka (4.3% and 2.7%, respectively). In the endorhizosphere fraction of rice, bacterial populations were very low. Fluorescent bacteria were absent.Based on 33 phenotypical tests, 89 fluorescent isolates were grouped into 5 clusters. The three major clusters covered the isolates belonging to the Pseudomonas fluorescens-putida group, whereas the remaining small clusters contained other UV-fluorescent bacteria. SDS-PAGE of total cell proteins enabled classification of the isolates into one of 12 different protein-polymorphic types. Only a partial correlation was found between the latter classification and the phenotypical one. Cyanogenesis was observed with strains of P. fluorescens only. Isolates P. fluorescens RW9S1 and P. cepacia RW5P1 displayed a potent antagonism against several fungi.     

Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt and rhizosphere colonization in pot-grown banana plantlets

Streptomyces sp. strain g10 exhibited strong antagonism towards Fusarium oxysporum f.sp. cubense (Foc) races 1, 2 and 4 in plate assays by producing extracellular antifungal metabolites. Treating the planting hole and roots of 4-week-old tissue-culture-derived Novaria banana plantlets with strain g10 suspension (10⁸ cfu/ml), significantly (P<0.05) reduced wilt severity when the plantlets were inoculated with 10⁴ spores/ml Foc race 4. The final disease severity index for leaf symptom (LSI) and rhizome discoloration (RDI) was reduced about 47 and 53%, respectively, in strain g10-treated plantlets compared to untreated plantlets. Reduction in disease incidence was not significant (P<0.05) when plantlets were inoculated with a higher concentration (10⁶ spores/ml) of Foc race 4. Rhizosphere population of strain g10 showed significant (P=0.05) increase of more than 2-fold at the end of the 3rd week compared to the 2nd week after soil amendment with the antagonist. Although the level dropped, the rhizosphere population at the end of the 6th week was still nearly 2-fold higher than the level detected after 2 weeks. In contrast, the root-free population declined significantly (P=0.05), nearly 4-fold after 6 weeks when compared to the level detected after 2 weeks. Neither growth-inhibiting nor growth-stimulating effects were observed in plantlets grown in strain g10-amended soil.     

T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment

The following study was undertaken to better understand the mechanisms that relate the homeostatic set point of the peripheral T cell population to energy availability in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+T cells notably declined after one week of malnourishment, a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Overall levels of IL-7 were similar in malnourished and control mice. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to quickly adjust the total number of T cells during malnutrition.     

Rhizosphere microflora ofCajanus cajan in relation to Fusarium wilt resistance

Summary Studies were made to find out the factors responsible for resistance inCajanus cajan (L.) Millsp. variety C-11-6 against wilt caused byFusarium udum Butl. It was thought to be either due to associated antagonistic microflora in the rhizosphere or due to biochemical constituents in the plant itself. Screening of the rhizosphere isolates revealed the absence of any potent antagonists againstF. udum suggesting that the resistance in C-11-6 variety is not due to antagonistic organisms in the rhizosphere.