Abhinandan Deora

Suppression of Damping-Off Disease in Host Plants by the Rhizoplane Bacterium Lysobacter sp. Strain SB-K88 Is Linked to Plant Colonization and Antibiosis against Soilborne Peronosporomycetes

ABSTRACT We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 μg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.

Isolation and Identification of Potential Phosphate Solubilizing Bacteria from the Rhizoplane of Oryza sativa L. cv. BR29 of Bangladesh

A total of 30 bacteria were isolated from the rhizoplane of rice cv. BR29 cultivated in Mymensingh, Bangladesh and from the seedlings obtained from surface-sterilized seeds of BR29. Upon screening, 6 isolates showed varying levels of phosphate solubilizing activity in both agar plate and broth assays using National Botanical Research Institute’s phosphate medium. The bacterial isolates were identified based on their phenotypic and 16S rRNA genes sequencing data as Acinetobacter sp. BR-12, Klebsiella sp. BR-15, Acinetobacter sp. BR-25, Enterobacter sp. BR-26, Microbacterium sp. BRS-1 and Pseudomonas sp. BRS-2. The BR-25 exhibited highest phosphate solubilizing activity followed by BR-15. They grew rapidly in the liquid medium at pH 5 and 7 but almost no growth occurred at pH 3. The pH value of the culture medium was decreased with bacterial growth suggesting that they might secrete organic acids to solubilize insoluble phosphorus. Scanning electron microscope analysis of two-week-old rice seedlings germinated from seeds previously inoculated with BR-25 and BR-15 revealed dense colonization at the root surfaces presumably using fimbriae on the bacterial cells.

An antagonistic rhizoplane bacterium Pseudomonas sp. strain EC-S101 physiologically stresses a spinach root rot pathogen Aphanomyces cochlioides

We observed that an antagonistic rhizoplane bacterium Pseudomonas sp. strain EC-S101 induces excessive lateral and apical branching in the hyphae of a root rot phytopathogen Aphanomyces cochlioides AC-5 resulting in radial growth inhibition of hyphae in a dual culture assay. Confocal laser scanning microscopic observations using fluorescent stains indicated an increased quantity of nuclei and lipid bodies in the affected hyphae during the early stage (less affected hyphae) at day 3 of interaction. At a more advanced stage (severely affected hyphae) at day 3, nuclei became smaller and round-shaped compared with the oval shape in AC-5 control hyphae. After 7 days, nuclei disintegrated, and the nuclear materials were released into the disorganized cytoplasm. With transmission electron microscopy at 5 days of interaction, we found that the cell walls of AC-5 hyphae were considerably thicker than those of the control. Enlarged vacuoles, lipid bodies sunk into vacuoles, and vacuoles filled with electron-dense material, followed by an invagination of the AC-5 hyphal cell wall, were commonly observed. Nonmembranous electron-transparent inclusion bodies irregular in size were often distributed in the affected hyphae. By integrating our observations, we conclude that antagonistic effects evoked by strain EC-S101 resulted in the death of AC-5 hyphae, which might contribute to the suppression of A. cochlioides AC-5-linked damping-off disease in its host plants.

Isolation and Structure Elucidation of Peronosporomycetes Hyphal Branching-Inducing Factors Produced by Pseudomonas jessenii EC-S101

Pseudomonas jessenii EC-S101 produced hyphal branching-inducing and mitosis-accelerating factors active towards Peronosporomycetes, Aphanomyces cochlioides hyphae. In searching for the active substances, EtOAc-solubles extracted from EC-S101-cultured solid medium were fractionated under the guidance of a paper disc assay using an A. cochlioides mycelium. Two active substances were subsequently isolated and the structure was elucidated by spectroscopic analysis to be (+)-4,5-didehydroacaterin (1) and 3-[(1R)-hydroxyhexyl]-5-methylene-2(5H)-furanone (2), both of which accelerated the mitotic process of A. cochlioides hyphae along with excessive branching at 1.0 μg per disc. These compounds are likely to affect the morphophysiological development of certain eukaryotic organisms in the terrestrial ecosystem.

Antagonistic effects of Pseudomonas jessenii against Pythium aphanidermatum: morphological, ultrastructural and cytochemical aspects

Pseudomonas jessenii isolate EC‐S101, an antagonistic rhizobacterium, induces morphological abnormalities such as topical swelling and excessive lateral branching in phytopathogenic Peronosporomycetes Pythium aphanidermatum hyphae as a result of radial growth inhibition in a dual culture assay. Rhodamine‐phalloidin staining revealed that these abnormalities were associated with disorganization of actin cytoskeleton. Both the morphological forms of actin, filaments and plaques, were affected progressively. At early stage of interaction (in less affected hyphae), the filaments were either eliminated or disarrayed. At advance stage of interaction (in severely affected hyphae), even the plaques population was decreased or disappeared. The effects of P. jessenii on actin architecture of Py. aphanidermatum were comparable to latrunculin B, a known actin assembly inhibitor. In addition, at early stage of interaction, the quantities of nuclei, lipid bodies and mitochondria became higher than those in control. At advance stage of interaction, the quantities of these organelles were almost similar, higher and lower, respectively, compare to those in control. Scanning electron microscopy exhibited cell wall disruption and accumulation of extracellular material associated with severely affected hyphae. Ultrastructural observations of the affected hyphae displayed additional features of considerable thickening of cell wall, enlargement of vacuoles, sinking of redundant lipid bodies into the enlarged vacuoles and wall appositions. We conclude that in addition to interference in morphogenesis and growth of Py. aphanidermatum, P. jessenii suppresses the pathogen through sub‐cellular disorganization, specifically the actin architecture. This is the first report on disruption of cytoskeleton in a eukaryotic phytopathogen by an antagonistic rhizobacterium. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes.

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 x 10(-6)m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.