Marshall S. Bergen

Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize

Endophytes are mutualistic symbionts within healthy plant tissues. In this study we isolated Bacillus spp. from seeds of several varieties of maize. Bacillus amyloliquifaciens or Bacillus subtilis were found to be present in all maize varieties examined in this study. To determine whether bacteria may produce antifungal compounds, generally lipopeptides in Bacillus spp., bacterial cultures were screened for production of lipopeptides. Lipopeptides were extracted by acid precipitation from liquid cultures of Bacillus spp. Lipopeptide extracts from Bacillus spp. isolated from Indian popcorn and yellow dent corn showed inhibitory activity against Fusarium moniliforme at 500μg per disk. Using MALDI-TOF mass spectrometry we detected the presence of antifungal iturin A, fengycin and bacillomycin in these isolates. PCR amplification also showed the presence of genes for iturin A and fengycin. B. subtilis (SG_JW.03) isolated from Indian popcorn showed strong inhibition of Arabidopsis seed mycoflora and enhanced seedling growth. We tested for the induction of defence gene expression in the host plant after treatment of plants with B. subtilis (SG_JW.03) and its lipopeptide extract using RT-qPCR. Roots of Indian popcorn seedlings treated with a suspension of B. subtilis (SG_JW.03) showed the induction of pathogenesis-related genes, including PR-1 and PR-4, which relate to plant defence against fungal pathogens. The lipopeptide extract alone did not increase the expression of these pathogenesis-related genes. Based on our study of maize endophytes, we hypothesize that, bacterial endophytes that naturally occur in many maize varieties may function to protect hosts by secreting antifungal lipopeptides that inhibit pathogens as well as inducing the up-regulation of pathogenesis-related genes of host plants (systemic acquired resistance).

Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession

Culturable fungal population diversity and succession was investigated in developing cranberry ovaries of fruit rot-resistant and rot-susceptible cranberry selections, from flower through mature fruit. Fungi were recovered in culture from 1185 of 1338 ovary tissues collected from June to September, yielding 2255 isolates that represented 42 morphotaxa. During the season, species richness varied from 2 to 17 and 2 to 18 in rot-resistant and rot-susceptible selections, respectively, increasing from wk1 to wk10 and then gradually declining to wk14. Shannon-Wiener diversity index varied from 0.27 to 2.32 in rot-resistant and 0.18 to 2.38 in rot-susceptible, and Pielou’s evenness index varied from 0.11 to 0.63 and 0.06 to 0.64 in rot-resistant and rot-susceptible selections, respectively, confirming that diversity of fungi in developing ovaries was similar among rot-resistant and rot-susceptible selections, but varied among sampling time points. Principal component analysis grouped samples collected at the same sampling time point together regardless of rot susceptibility of the selections, and detected the predominant fungal species associated with each stage of development. Successional changes were observed in populations of endophytic, pathogenic and saprophytic fungi throughout the season as ovaries matured.

A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria

In this paper we propose and provide evidence for a mechanism, oxidative nitrogen scavenging (ONS), whereby seedlings of some grass species may extract nitrogen from symbiotic diazotrophic bacteria through oxidation by plant-secreted reactive oxygen species (ROS). Experiments on this proposed mechanism employ tall fescue (Festuca arundinaceae) seedlings to elucidate features of the oxidative mechanism. We employed 15N2 gas assimilation experiments to demonstrate nitrogen fixation, direct microscopic visualization of bacteria on seedling surfaces to visualize the bacterial oxidation process, reactive oxygen probes to test for the presence of H2O2 and cultural experiments to assess conditions under which H2O2 is secreted by seedlings. We also made surveys of the seedlings of several grass species to assess the distribution of the phenomenon of microbial oxidation in the Poaceae. Key elements of the proposed mechanism for nitrogen acquisition in seedlings include: 1) diazotrophic bacteria are vectored on or within seeds; 2) at seed germination bacteria colonize seedling roots and shoots; 3) seedling tissues secrete ROS onto bacteria; 4) bacterial cell walls, membranes, nucleic acids, proteins and other biological molecules are oxidized; 5) nitrates and/or smaller fragments of organic nitrogen-containing molecules resulting from oxidation may be absorbed by seedling tissues and larger peptide fragments may be further processed by secreted or cell wall plant proteases until they are small enough for transport into cells. Hydrogen peroxide secretion from seedling roots and bacterial oxidation was observed in several species in subfamily Pooideae where seeds possessed adherent paleas and lemmas, but was not seen in grasses that lacked this feature or long-cultivated crop species.

An examination of molecular phylogeny and morphology of the grass endophyte Balansia claviceps and similar species

A molecular and morphological study is made of several species of Balansia, including Bal- ansia andropogonis, B. asclerotiaca, and B. asperata, that are often confused with Balansia claviceps, the type species of genus Balansia. Of these species, only B. claviceps is endophytic and possesses mounds of long receptive hyphae. Both morphology and rDNA data suggest that the collections currently recognized as B. claviceps consist of multiple sibling species. DNA data demonstrate the presence of two clades in Bal- ansia, denominated by the B. claviceps and B. asper- ata clades. The distribution of species native to Asia and the Americas among these two subclades sug- gests the movement of species between Asia and the Americas.

Features and phylogenetic status of an enigmatic clavicipitalean fungus Neoclaviceps monostipa gen. et sp. nov.

A new species and genus of the gramini- colous Clavicipitaceae are described. Neoclaviceps monostipa infects individual florets in the same way as species of genus Claviceps but does not produce sclerotia. In culture it shows dimorphism in produc- tion of a yeast-like microcyclic conidiation (ephelidi- al) phase and a mycelial phase. Phylogenetic analysis of the 26S large subunit rDNA suggests that Neoclav- iceps along with genus Myriogenospora are evolution- arily intermediate between Claviceps and Balansia.

Occurrence of Bacillus amyloliquefaciens as a systemic endophyte of vanilla orchids

We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids (Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia × V. pompona) as a systemic bacterial endophyte. We determined with light microscopy and isolations that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial endophyte and that shoot meristems and stomatal areas of stems and leaves were densely colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence data. Since additional endophyte‐free plants and seed of this orchid were not available, additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea tricolor, and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens demonstrated intracellular colonization of guard cells and other epidermal cells, confirming the pattern observed in the orchids. Isolations and histological studies suggest that the bacterium may penetrate deeply into developing plant tissues in shoot meristems, forming endospores in maturing tissues. B. amyloliquefaciens produced fungal inhibitors in culture. In controlled experiments using morning glory seedlings we showed that the bacterium promoted seedling growth and reduced seedling necrosis due to pathogens. We detected the gene for phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of antifungal lipopeptides, and purified the lipopeptide “surfactin” from cultures of the bacterium. We hypothesize that B. amyloliquefaciens is a robust endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between this bacterium and its hosts can be managed to protect vanilla crops from diseases is a question that should be evaluated in future research. Microsc. Res. Tech. 77:874–885, 2014.

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′‐diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed‐transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H2O2 staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H2O2 staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech. 77:566–573, 2014.

Induction of salt tolerance and up-regulation of aquaporin genes in tropical corn by rhizobacterium Pantoea agglomerans

Bacteria were isolated from surface disinfected seeds of eight modern corn types and an ancestor of corn, ‘teosinte’ and identified using 16S rDNA sequences. From each of the modern corn types we obtained Bacillus spp. (including, Bacillus amyloliquefaciens and Bacillus subtilis); while from teosinte we obtained only Pantoea agglomerans and Agrobacterium species. Of these bacteria, only P. agglomerans could actively grow under hypersaline conditions and increase salt tolerance of tropical corn seedlings. In laboratory and greenhouse experiments where plants were watered with a 0·2 mol l−1 NaCl solution, P. agglomerans was found to enhance the capacity of tropical corn to grow compared to uninoculated controls. The total dry biomass was significantly higher in P. agglomerans‐treated plants compared to controls under saline water. Gene expression analysis showed the up‐regulation of the aquaporin gene family especially plasma membrane integral protein (ZmPIP) genes in P. agglomerans‐treated plants. The plasma membrane integral protein type 2 (PIP2‐1) gene in tropical corn seedlings was highly up‐regulated by P. agglomerans treatment under salt stress conditions. Microscopic examination of P. agglomerans inoculated seedlings revealed that the bacterium colonized root meristems densely, and as roots developed, the bacterium became sparsely located in cell junctions.

Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils

This article describes a process (termed ‘oxidative nitrogen scavenging’) where grasses scavenge organic nitrogen from microbes on and around roots. The authors propose a diurnal process where during the day roots produce and release hydrogen peroxide that oxidizes microbial exoenzymes around roots; at night hydrogen peroxide production ceases, then roots and symbiotic rhizobacteria secrete proteases that degrade the oxidized proteins to form peptides that are absorbed by roots. The existence of a mechanism for organic nitrogen scavenging in grasses emphasizes the nutritional importance of non-pathogenic microbes that associate with roots. Future applications of this process could result in new methods for the cultivation of crop plants.

Functional role of an endophytic Bacillus amyloliquefaciens in enhancing growth and disease protection of invasive English ivy (Hedera helix L.)

BackgroundWe hypothesize that invasive English ivy (Hedera helix) harbors endophytic microbes that promote plant growth and survival. To evaluate this hypothesis, we examined endophytic bacteria in English ivy and evaluated effects on the host plant.MethodsEndophytic bacteria were isolated from multiple populations of English ivy in New Brunswick, NJ. Bacteria were identified as a single species Bacillus amyloliquefaciens. One strain of B. amyloliquefaciens, strain C6c, was characterized for indoleacetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis against pathogens. PCR was used to amplify lipopeptide genes and their secretion into culture media was detected by MALDI-TOF mass spectrometry. Capability to promote growth of English ivy was evaluated in greenhouse experiments. The capacity of C6c to protect plants from disease was evaluated by exposing B+ (bacterium inoculated) and B− (non-inoculated) plants to the necrotrophic pathogen Alternaria tenuissima.ResultsB. amyloliquefaciens C6c systemically colonized leaves, petioles, and seeds of English ivy. C6c synthesized IAA and inhibited plant pathogens. MALDI-TOF mass spectrometry analysis revealed secretion of antifungal lipopeptides surfactin, iturin, bacillomycin, and fengycin. C6c promoted the growth of English ivy in low and high soil nitrogen conditions. This endophytic bacterium efficiently controlled disease caused by Alternaria tenuissima.ConclusionsThis study suggests that B. amyloliquefaciens plays an important role in enhancing growth and disease protection of English ivy.