Sunagoke Moss (Racomitrium japonicum) Used for Greening Roofs Is Severely Damaged by Sclerotium delphinii and Protected by a Putative Bacillus amyloliquefaciens Isolate

Mosses are ecologically important plants also used for greening, gardening, and decorative purposes. Knowledge of the microbial flora associated with mosses is expected to be important for control and preservation of global and local environments. However, the moss-associated microbial flora is often poorly known. Moss-associated fungi and bacteria may promote plant growth and pest control, but they may be alternative hosts for pathogens of vascular plants. In this study, the fungus Sclerotinia delphinii was identified for the first time as a pathogen that causes severe damage to Sunagoke moss (Racomitrium japonicum). This moss is used for greening roofs and walls of buildings in urban environments owing to its notable tolerance of environmental stresses. Inoculation with the S. delphinii strain SR1 of the mono- and dicotyledonous seed plants Hordeum vulgare, Brassica rapa var. pekinensis, Lactuca sativa, and Spinacia oleracea, in addition to the liverwort Marchantia polymorpha and the moss Physcomitrella patens, showed that the fungus has a wide host range. Colonization with SR1 progressed more rapidly in non-vascular than in vascular plant species. Studies with P. patens under controlled conditions showed that SR1 secreted a fluid during colonization. Treatment with the secretion induced production of reactive oxygen species in the moss. Endogenous peroxidase partially inhibited SR1 colonization of P. patens. A bacterial isolate, most likely Bacillus amyloliquefaciens, that coexists with R. japonicum was antagonistic to SR1 growth. Taken together, the present results suggest that fungal colonization of mosses may be prevented by a peroxidase secreted by the moss and an antagonistic bacterium coexisting in the moss habitat. The findings suggest that there is potential to apply biological control measures for protection of mosses against fungal pathogens.Peer reviewe

Physcomitrella patens Has Kinase-LRR R Gene Homologs and Interacting Proteins

Peer reviewe

Practical Application of Methanol-Mediated Mutualistic Symbiosis between Methylobacterium Species and a Roof Greening Moss, Racomitrium japonicum

Bryophytes, or mosses, are considered the most maintenance-free materials for roof greening. Racomitrium species are most often used due to their high tolerance to desiccation. Because they grow slowly, a technology for forcing their growth is desired. We succeeded in the efficient production of R. japonicum in liquid culture. The structure of the microbial community is crucial to stabilize the culture. A culture-independent technique revealed that the cultures contain methylotrophic bacteria. Using yeast cells that fluoresce in the presence of methanol, methanol emission from the moss was confirmed, suggesting that it is an important carbon and energy source for the bacteria. We isolated Methylobacterium species from the liquid culture and studied their characteristics. The isolates were able to strongly promote the growth of some mosses including R. japonicum and seed plants, but the plant-microbe combination was important, since growth promotion was not uniform across species. One of the isolates, strain 22A, was cultivated with R. japonicum in liquid culture and in a field experiment, resulting in strong growth promotion. Mutualistic symbiosis can thus be utilized for industrial moss production

Growth promotion of seed plants by added <i>Methylobacterium</i> species.

<p>Data presented as mean ± SD. Fresh mass indicates that of entire plants.</p><p>nt: not tested.</p>*<p>, p<0.05 and.</p>**<p>, p<0.01 (Student's T-test).</p

Growth promotion of <i>R. japonicum</i> by <i>Methylobacterium</i> isolates.

<p>A, Protonemata growth on Y medium in the presence of bacterial isolates (35 days of cultivation). Grid, 5 mm. B, Quantification of protonemata growth. White bars, 22 days; light gray bars, 35 days; and gray bars, 63 days. Growth was evaluated by measuring the area of protonemata proliferation, regarded as ellipses. Error bars, standard deviation (n = 8). C, Strain 22A growing with protonemata on Y medium. Grid, 5 mm. D, Induction of gametophyte formation by application of bacterial isolates. A suspension of <i>R. japonicum</i> protonemata was inoculated on Florialite, to which 50 µL of water (control), kinetin (3 mg/L), or a bacterial suspension (11A, 21C and 22A) was applied. Images were taken after 60 days of cultivation.</p

Result of DGGE analysis for <i>R. japonicum</i> liquid culture samples.

<p>Identification was done at EZtaxon site (version 2.1). The closest relatives are listed.</p

Detection of methanol emitted from <i>R. japonicum.</i>

<p>A. Control observation with <i>P. pastoris</i> PPY12 (pYA005 and pYA006) grown on YNB and YNM for 2 days. B. YNB-grown cells were applied onto Y medium, Y+0.5% methanol (YM); protonemata were grown on Y medium and non-sterile gametophytes were inoculated on Y medium. Left panel, fluorescence; right panel, differential interference contrast images. Bar, 20 µm.</p

DGGE profiles showing the bacterial communities of <i>R. japonicum</i> liquid cultures.

<p>A–D indicate the growth conditions (see text) and 1, 4 and 7 refer to the week of sampling. Bands indicated with numbers were purified and sequenced (for results, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033800#pone-0033800-t001" target="_blank">Table 1</a>).</p