1 Epiphytic bacteria on lichens

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Biochemical Responses of Plants to Invaders, 2008: 1-11 ISBN: 978-81-308-0306-7 Editors: María Estrella Legaz and Carlos Vicente

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Epiphytic bacteria on lichens Eva-María Díaz, Susana Rodríguez and Julia Quintana Department of Plant Biology I, Faculty of Biology, Complutense University Madrid, Spain

Introduction Bacteria of many species inhabit the leaf surface of plants and show a high rate of growth. Then, they are often considered as the primary invaders. Many phylloplane bacteria contain carotenoids. This has been interpreted as a defense mechanism against high light intensities. Phylloplane bacteria are able to use wax and cutin as carbon sources and thus the wax appears dissolved in their vicinity. Other, as Leuconostoc mesenteroides or Erwinia amylovora, are able to use small amounts of sucrose secreted by sugarcane leaves but they are unable to enter inside the leaf [1]. Some cyanobacteria, such as Nostoc or Correspondence/Reprint request: Dr. Eva-María Díaz, Department of Plant Biology I, Faculty of Biology Complutense University, Madrid, Spain. E-mail: [email protected]

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Eva-María Díaz et al.

Anabaena are nitrogen-fixing microorganisms and they currently occur on leaves [2]. Bacteria producing ice nuclei cause water freezing at relatively moderate temperature values, around –2.3°C, whereas pure water can be frozen at – 15°C before complete freezing in the absence of ice nuclei. This implies that bacteria are able to minimize the damage produced by frosts by inducing the formation of ice crystals at not excessively low temperature values, with an extracellular location. Brush et al [3] found that the maximal activity of ice nucleation in leaves of Secale cereale was achieved at –8°C or –9°C whereas that of their epiphytic bacteria, living on the leaves, was displaced towards – 12°C, temperature at which ice nucleation in leaf tissues decreased at the minimum quantitatively valuable. The formation of these extracellular, crystalline ice nuclei protects the cell against the loss of cytoplasmic heat and their eventual liquefaction provided a gain of liquid water to the cell.

Epiphytic bacteria on lichen thallus Currently we have acquired a great knowledge about the symbiotic components of lichens but there is no enough information about the microbial communities in the lichen environment. Lythic substrate, which many lichens colonize, is occupied by a large spectrum of microorganisms that compose lythobiontic communities. Microorganisms on lichen thalli and other epiphytic, endolythic or free-living organisms can be distinguished. Under the lichen thallus, these relationships are much more intimate, forming a complex biofilm between the lower surface of the lichen and the upper surface of stone. In this interfacies, thalli-stone substrate, many biophysical and biochemical processes are achieved, producing substrate alterations and the emergence of microenvironments where very peculiar lythobiontic communities develop. Both fungi on lichens and fungi-forming lichens secrete many organic compounds that are absolutely required to supply mineral nutrients from the substrate to the lichen thallus [4,5,6]. In other words, organic acids from fungi and lichens are important for the solubilization of inorganic substances constitutive of mineral substrates.

Microbial alterations of lichen substrata An ultrastructural study of the interfacies thallus-substrate of Lasallia hispanica, Parmelia omphalodes and Cornicularia normoerica, three saxicolous lichen species growing on granite, was carried out by de los Ríos et al. [7]. In these zones, lythobiontic communities composed by both foliose and fruticose lichens, crustaceous lichens and free-living fungi and algae develop, producing physical and chemical alterations of the substrate.

Epiphytic bacteria on lichens

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Anchorage elements of the thallus to the substrate produce cracks and fissures that are colonized by endolythic microorganisms. On the other hand, the mineral transformation of the substrate was also detected, being mainly potassium biomobilized in some extent. Biofilm production is a major characteristic of the microbial growth in nature. These biofilms have been observed in many different environments, although the process of mineral extraction from the substrate is poorly known. Biofilm properties often change depending on the functional and ultrastructural inter-relationships between their components [8]. Actions of these biofilms can be divided into two main components, one of physical nature through erosion and fragmentation of the stone, derived from the growth of the biofilm itself and through the variation in water disposal, and another one, of chemical nature, through solution and chelation processes favoring the formation of metallic complexes with metabolic products of the biofilm [9], as well as with polymeric, extracellular substances. In addition, biomobilization actions carried out by lichens and cyanobacteria, and biomineralization frequently effected by cyanobacteria and microorganisms, are also achieved. These processes, together clay and quartz deposition and retention of toxic contaminant agents, are involved in the degradation of monumental stones [8,10,11].

Epiphytic microorganisms collaborate to the supply of nutrients to lichens Recent studies [12] found that heterotrophic, N2-fixing microorganisms colonize fungi in common soils through biofilm production. Moreover, the occurrence of N2-fixing organisms in biofilms stimulates the production of organic acids [13]. The variability of organic acids produced can be due to the specific union bacteria-fungi, that controls their interactions as well as the quality/quantity of the organic acids secreted to the medium [14]. Microbiological unions are very important in order to favor the mineral alteration of the substrate [15]. Cardinale et al. [16] realized the molecular characterization of bacterial communities associated to some lichen species, Cladonia spp., Pseudevernia furfuracea, Hypogymnia physodes and Rocella sp., collected from different habitats. Thirty-four morphological types of bacteria, 18 epiphytes and 16 intrathalline forms, were isolated. Phenotypes were purified and grouped in 25 phenotypes. At genre level, 3 from Firmicutes, 4 from Actinobacteria, and 5 from Proteobacteria were identified. Association of Paenibacillus and Burkholderia to lichens was not surprising since the existence of association of these genera to different fungi is well documented. However, species from

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Azotobacter were not detected although these bacteria had been isolated from other lichen species [17]. Neither Clostridium nor Beijerickia or Pseudomonas was found. Composition of bacterial communities associated to lichens was usually affected by both abiotic and biotic factors, including the phyllogenetical position of lichens, their geographical origin, substrate, conditions of lichen-produced microhabitat and secondary metabolites produced by these lichens. González et al. [18] studied the diversity of ascomycetes population isolated from lichens collected from tropical areas of Hawaii, Reunion Islands and cold areas of Alaska. Actinomycetes are a well-known group of filamentous, Gram positive bacteria. They are widespread in soils and play and important role in the nutrient cycle. They are able to establish saprophytic relationships and to use all sources of nutrient to grow. They produce many secondary metabolites involved in the maintenance, signaling and colonization [19,20]. Through the analysis of the composition of fatty acids and other molecular methods, the diversity of actinomycetes inhabiting lichens has been studied. By using PCR, genes associated to the production of secondary metabolites have been analyzed to evaluate the biosynthetic potential of these microorganisms [21,22,23] and latter, it has been compared to their antimicrobial activity by isolation in laboratory conditions. A major diversity of microorganisms has been detected in lichens from hot rather than cold environments. The nature of the substrate does not affect microbial diversity. A low antimicrobial activity can reflect the loss of expression of some biosynthetic systems in the probe conditions or its relationship with biosynthetic processes different from that conducting to the production of active substances. As a conclusion, lichens constitute a very interesting source for the isolation of a wide variety of actinomycetes, which are an important reserve of secondary metabolites. Results obtained as yet indicate that actinomycetes are not merely saprophytic organisms but a pool of biosynthetic pathways passively transferred through the different member of the microbial ecological niche.

Cryptoendolythic communities Cryptoendolythic communities are composed by microorganisms that colonize stone pores. These communities occur around the world and they play a role in the global process of rock alteration and nutrient cycling. However, our knowledge about their composition and dynamic is poor. These communities have been characterized by microscopic analysis and culture studies whereas phylogenetic analyses are scanty [24]. One of the main


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problems is that many of these microorganisms survive as undetectable forms [25]. Thus, the composition and biochemical activities of those cannot be extrapolated only from microbiological analyses. One of the best-known examples is the endolythic community of the polar desert of McMurdo Valley (Antarctic) where these populations are the main form of life. De la Torre et al. [26] found that lichens and cyanobaceria are the dominant forms in the community. Unfortunately, comparison of molecular analysis results and microscopic observations is very difficult because of the lack of sequencing studies of the majority of the organisms previously identified by morphological analysis. In spite of this disadvantage, microbial communities of the Antarctic coast are dominated by bacteria, cyanobacteria, fungi and algae. Thin layers of mushrooms and lichens cover soils with a few cm in thickness, which accumulate high amounts of sodium chloride from the sea spray. Strong winds and cryoclasia processes form fine-powdered materials, which accumulate at lee of the rolling stones, generating microclimate conditions of wetting and temperature. Between rock, new different microenvironments are established: mineral soils, mushrooms and lichen communities of the soil, fruticose terricolous lichens, etc. [27,28]. Thus, in few cm, environmental conditions are very different for microhabitats with different associations plant-microorganisms. Studies achieved by Bölter [29] show that very hard conditions of temperature and wetting of the Antarctic coast must be considered as stress factors that cause microorganism selection with a great ecological wide. On the determinant elements for the microbial community is the availability of minimize its metabolic activity under stress. A high metabolic rate and a low level of organic matter indicate a fine relationship between photoergonic and chemoergonic organisms. Low molecular weight carbohydrates can attain very high values of concentration because of lichens and moss lixiviation. However, microorganisms can use this material during short time periods, especially during the spring thaw. Relief and topography of the Antarctic coast have been controlled by microclimate factors as well as by plant development. These facts can be considered as the basis for the establishment of microzones in which the biological activity is controlled by short periods of favoring temperature and wetting as well as by the disposal of sufficient organic matter.

Changes in lichen metabolism produced by epiphytic bacteria In the lichen symbiosis, the fungus establishes a symbiotic relationship with a green alga or with a cyanobacterium. In this mutualistic association,

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the mycobiont produces an adequate environment for the survival of the phyco- (or cyano-) biont, favoring gas exchange between the thallus and the atmosphere, supplying water and nutrient and protecting it against desiccation. The algal partner, in turn, supplies to the mycobiont with organic matter through photosynthesis and the cyanobacterial partner supplies reduced nitrogen compounds from atmospheric dinitrogen [30]. By considering that lichens habitually live under severe environmental conditions and low nutrient disposal, the occurrence of heterotrophic bacteria as a secondary partner in the association provides enormous advantages for both myco- and photobionts. In cyanobacteria, the occurrence of epiphytic bacteria on lichen thallus, interaction with the mycobiont for the production and/or accumulation of phenolics compounds has been found by Blanco et al. [31]. On the other hand, chemoorganotrophic, nitrogen-fixing bacteria are able to produce and secrete reduced nitrogen compounds, such as amino acids, vitamins [32] and phytohormones [33,34]. This fact results particularly important when the study of the possible interaction between bacteria and lichens is attempted. Liba et al. [35] studied the occurrence of nitrogen-fixing, chemoorganotrophic bacteria in five lichen species, Canoparmelia caroliniana, C. crozalsiana, C. texana, Parmotrema sancti-angeli and P. tinctorum. From these species, Stenotrophomonas maltophilia, Pseudomonas spp., Pantoea spp., Serratia marcescens and Acinetobacter calcoaceticus were isolated. All these bacteria were able to secrete amino acids, about 64% of these were able to solubilized phosphate, and about 30% produced ethylene. No specificity in the bacteria-lichen association has been found. As yet, no studies about the role of epiphytic bacteria on the activity of lichen cells have been performed although they have been found in several lichen systems. However, when glucose instead of acetate was supplied to immobilize cells of Cladonia substellata, catabolite repression prevents usnic acid production. In addition, proteobacteria rapidly develop and disaggregate immobilisates [37]. The production of phenolics by immobilized cells of Pseudevernia furfuracea loaded with penicillin more than in non-treated bioreactors. One explanation for this could be that lichen cells used some cofactors provided from bacterial metabolism to synthesize atranorin and physodic acid, the main phenols produced by this lichen species. Production of atranorin required two consecutive reaction depending on NAD+ and NADH, respectively, whereas the synthesis of physodic acid needed a supply on an acyl-CoA to introduce a CH3-(CH2)5-CO- at the C6 position. A critical bacterial biomass could be required to supply some of these cofactors, as the final increase of the production of phenols suggest [37].

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Alterations by lichens and their associated bacteria of monumental stones The erosion is not the unique negative effect of lichens on monumental stones: Sometimes, changes in the colour of the rock produce undesirable pigmentation. Milk-like films of calcium oxalate or the darkening of the stone surface are the main symptoms of these biological activities. Microbiological analysis of stone surfaces of San Juan del Duero (Soria, Spain) Cloister revealed the occurrence of lichen-associated sulfobacteria responsible of the biological reduction of sulfates contained by the lichen substrate, according to the sequenced reactions:

ATP ATP + SO 42-

ADP

APS

PAPS

S2-

SO326e-

Figure 1. Stiple from San Juan del Duero Cloister showing black deposits (arrows) of lead sulfure produced by sulfate reduction achieved by lichen-associated bacteria.

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Black warehouse were produced by deposition of insoluble lead sulfure in cracks and pores of the stone (Fig. 1). The origin of lead was undoubtedly the aerosol produced by the combustion of car fuels. To know the nature of the external electron donor in the reduction process, an experiment was carried out using ground stone from the quarry near the Cloister mixed with disaggregate lichen thalli. This mixture was rehydrated with the adequate buffer containing 10 µM lead chloride and maintained for several days at 30°C in light or in the dark. A control in which the lichen cell suspension was previously autoclaved was maintained in the same conditions. Powdered stone only was stained by lead sulfure when the mixtures were maintained in light. Thus, it can be concluded that sulfobacteria, epiphytes on the lichen thallus need lichen photoassimilates as a source of electrons to support the sulfite reductasa activity [36].

Primary characterization of lichen associated, sulfatereducing bacteria We attempt to obtain more information about bacteria from lichens able to reduce sulfates. Xanthoria parietina thalli, growing on granite or epiphytic on Platanus occidentalis, were recently collected, washed with sterile, distilled water and this wash was used to inoculate liquid media containing peptone, 5.0 g; sucrose, 20 g; sodium dihydrogen phosphate, 0.5 g; magnesium sulfate, 0.25 g; nicotinic acid, 0.01 g per liter. These media were maintained at 30°C in light or in the dark for 5 days. At the end of this time period, only media maintained in the light showed a substantial growth of microorganisms. Cells were collected by centrifugation at 10000 × g for 20 min at 2°C, washed with sterile, distilled water and separated in two samples. Cells obtained by culturing microorganisms from saxicolous lichen showed a clear purple colour whereas those obtained after culturing microorganisms from epiphytic lichen were white in colour. One of the samples was extracted with 80% acetone (v/v) in the dark for 24h. Cells were collected by centrifugation and then extracted with distilled water, disrupted in a sonic oscillator at 20 Hz s-1 for 2 min with ice protection, centrifuged at 14000 × g for 20 min at 2°C and the pellet was discarded. Absorption spectra of both acetonic and aqueous extracts were recorded from 350 nm to 1100 nm. Results in Fig. 2 indicated that both purple bacteria from saxicolous lichens and white bacteria from epiphytic lichens contained a pigment that absorbs at 969 nm, extracted with acetone, whereas only purple bacteria contained a water-soluble pigment showing an absorbance maximum at 416 nm and secondary maxima at 525 nm and 550 nm. This indicates the presence of a b-type cytochrome, which is characteristic of several species of Desulfotomaculum,

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A Bacteria from saxicolous lichen Bacteria from epiphytic lichen

B Bacteria from saxicolous lichen Bacteria from epiphytic lichen

Figure 2. Absorption spectra of an acetonic (A) or an aqueous (B) extract from bacteria isolated from Xanthoria parietina thallus.

a soil, Gram positive and anaerobic bacteria able to reduce sulfates to sulfure using organic compounds as reducing factors [38]. The second sample was used to inoculate powdered barite (barium sulfate) through three different applications. The first contained only bacteria, the second, bacteria mixed with a cell-disaggregated lichen thallus, and the third, only lichen cells. After 4h at 30°C in light or in the dark, only purple bacteria obtained from saxicolous lichen, maintained in light, were able to produce sulfure, revealed after treatment with 1% (w/v) lead chloride solution (Fig. 3). This confirms the previous hypothesis about the requirement of lichen metabolites as reducing compounds to the bacterial reduction of sulfate to sulfure.

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A

B

C

D

Figure 3. Combined actions if epiphytic bacteria on Xanthoria parietina thallus and lichens cells of sulfate reduction from barite. A) Bacteria from epiphytic Xanthoria in the dark. B) Bacteria from epiphytic Xanthoria in light C) Bacteria from saxicolous Xanthoria in the dark, D) Bacteria from saxicolous Xanthoria in light. 1 = Bacteria only; 2 = Bacteria and lichen cells; 3 = Lichen cells only.

Acknowledgements We gratefully acknowledge the excellent technical assistance of Rocío Santiago and the valuable suggestions of Prof. Carlos Vicente.

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