An ultrastructural investigation of the surface microbiota present on the leaves and reproductive structures of the resurrection plant Myrothamnus flabellifolia

The leaves, flower and stems of the southern African angiosperm resurrection plant Myrothamnus flabellifolia were investigated at the ultrastructural level to determine the source of previously reported fungal contamination. Fungal mycelia and hyphae of the genera Aspergillus and Penicillium were found localized to the hydathodes of the leaves and stigmatic surfaces of the female flowers in both desiccated and hydrated specimens. A waxy bacterium of the genus Bacillus was found to colonise the waxy epidermal surfaces of the leaves and flowers which was also where fungal cells were found to be absent. It is suggested that the wax like deposits within the leaves and stems as well as over the epidermal surface prevent the growth of the fungal organisms. These fungi opportunistically invade moist surfaces, such as the floral stigmas, during periods of moisture availability and may thus negatively impact plant development.     

Biominerals and waxes of <Emphasis Type="Italic">Calamagrostis epigejos</Emphasis> and <Emphasis Type="Italic">Phragmites australis</Emphasis> leaves from post-industrial habitats

Vascular plants are able to conduct biomineralization processes and collect synthesized compounds in their internal tissues or to deposit them on their epidermal surfaces. This mechanism protects the plant from fluctuations of nutrient levels caused by different levels of supply and demand for them. The biominerals reflect both the metabolic characteristics of a vascular plant species and the environmental conditions of the plant habitat. The SEM/EDX method was used to examine the surface and cross-sections of the Calamagrostis epigejos and Phragmites australis leaves from post-industrial habitats (coal and zinc spoil heaps). The results from this study have showed the presence of mineral objects on the surfaces of leaves of both grass species. The calcium oxalate crystals, amorphous calcium carbonate spheres, and different silica forms were also found in the inner tissues. The high variety of mineral forms in the individual plants of both species was shown. The waxes observed on the leaves of the studied plants might be the initializing factor for the crystalline forms and structures that are present. For the first time, wide range of crystal forms is presented for C. epigejos. The leaf samples of P. australis from the post-industrial areas showed an increased amount of mineral forms with the presence of sulfur.     

Ozone-induced ethylene release from leaf surfaces

Ozone-induced stress ethylene emissions from the adaxial and abaxial leaf surfaces of four plant species (Glycine max [L.] Merr. cv. Dare, Lycopersicon esculentum Mill cv. Roma VF, Eucalyptus globulus Labill. and Hedera helix L.) were studied to determine if the stress ethylene diffused through the stomata or cuticle. In plants not exposed to ozone, basal ethylene was detected above both the adaxial and abaxial leaf surfaces of all the plant species examined, indicating that some ethylene can diffuse across the leaf cuticle. Ozone induced stress ethylene production in all species examined. Significant ozone-induced ethylene concentrations were detected above both surfaces of amphistomatous soybean (Glycine) and tomato (Lycopersicon) leaves. In contrast, ozone-induced ethylene production was associated only with the leaf surface (abaxial) that contained stomata for hypostomatous blue gum eucalyptus and English ivy (Hedera) leaves; the leaf surface (adaxial) of the eucalyptus and ivy leaves which did not contain stomata did not release significant amounts of stress ethylene. These data indicate that ozone-induced stress ethylene primarily diffuses from the leaf via the stomata.     

风毛菊属3种植物叶的解剖结构比较

采用石蜡切片法对分布于祁连山海拔5 000 m左右流石滩上菊科风毛菊属水母雪兔子（Saussurea medusa Maxim）、鼠曲雪兔子（Saussurea gnaphalodes (Royle) Sch.）、红叶雪兔子（Saussurea paxiana Diels.）3种植物叶片的解剖结构进行了比较研究,结果表明：叶片表皮细胞均为单层,上下表皮都有气孔分布,气孔不下陷;角质层较厚,叶表面均被单列细胞的表皮毛。3种植物均为异面叶;叶肉栅栏组织较发达,通常由2～3层细胞组成,但栅栏组织细胞排列较疏松;海绵组织存在大量的细胞间隙;叶肉中通气组织发达,且均有不规则裂生性气腔。叶脉维管束中韧皮部都具有异细胞存在。这些共同特征是3种植物对高山地区缺氧、低温、强辐射等自然条件长期适应的结果。但是,3种植物在叶片的外部形态特征、叶肉栅栏组织细胞的特点、维管束发育程度、内分泌结构、不规则裂生气腔等方面又存在明显的不同,表现出3种植物对环境的适应也是存在差异的。     

Direct analysis of Peucedanum palustre samples by desorption atmospheric pressure photoionization-mass spectrometry

Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that can be used for the analysis of polar and nonpolar compounds directly from surfaces. Here, the feasibility of DAPPI-MS in the screening of plant metabolites from dried Peucedanum palustre leaves and umbels was studied. DAPPI-MS requires no prior sample preparation or chromatographic separation, and the analysis can therefore be performed directly from the untreated plant material. P. palustre contains several linear and angular furanocoumarins, some of which are specific for the species. The DAPPI mass spectra of both leaf and umbel samples showed distinct ions at m/z 445 and 443 in positive and negative ion modes, respectively. MS² analyses of these ions confirmed that the ions were the protonated and deprotonated molecules, respectively, of peulustrin and its isomers, which have only been identified from P. palustre. The direct analysis of dried plant material by DAPPI-MS was shown to provide a fast and reliable means to confirm the identity of plant materials, to study the metabolite profiles of plants, and to screen biologically relevant compounds from plant surfaces.     

Assessing the role of vertical leaves within the photosynthetic function of Styrax camporum under drought conditions

Previous evidence has demonstrated that vertical leaves of Styrax camporum, a woody shrub from the Brazilian savanna, have a higher net photosynthetic rate (P _N) compared with horizontal leaves, and that it is detected only if gas exchange is measured with light interception by both leaf surfaces. In the present study, leaf temperature (T _leaf), gas exchange and chlorophyll (Chl) a fluorescence with light interception on adaxial and also on abaxial surfaces of vertical and horizontal mature fully-expanded leaves subjected to water deficit (WD) were measured. Similar gas-exchange and fluorescence values were found when the leaves were measured with light interception on the respective surfaces of horizontal and vertical leaves. WD reduced P _N values measured with light interception on leaf surfaces of both leaf types, but the effective quantum yield of PSII (Φ_PSII) and the apparent electron transport rate (ETR) were reduced only when the leaves were measured with light interception on the adaxial surface. WD did not decrease the maximum quantum yield of PSII (F_v/F_m) or increase T _leaf, even at the peak of WD stress. Vertical leaf orientation in S. camporum is not related to leaf heat avoidance. In addition, the similar P _N values and the lack of higher values of Φ_PSII and ETR in vertical compared with horizontal leaves measured with light interception by each of the leaf surfaces suggests that the vertical leaf position is not related to photoprotection in this species, even when subjected to drought conditions. The exclusion of this photoprotective role could raise the alternative hypothesis that diverse leaf angles sustain whole plant light interception efficiency increased in this species.     

Geotaxis and leaf-surface preferences mitigate negative effects of a predatory mite on an herbivorous mite

Reproductive success and population growth of an herbivorous mite are limited by activities of phytoseiid predators. However, occurrences on upper versus lower leaf surfaces are sometimes mismatched between these prey and predators. The mismatch potentially mitigates predation risk for the prey species. We assessed factors that affect mite distributions on leaf surfaces, testing whether the presence of the phytoseiid mite Phytoseius nipponicus alters the leaf-surface distribution and reproductive success of the herbivorous false spider mite Brevipalpus obovatus. The host plant was Viburnum erosum var. punctatum (Adoxaceae). Leaves were set in natural (TRUE) and reversed (upside down; INVERTED) orientations using experimental devices. Both surfaces were accessible to mites. We detected lower and abaxial leaf-surface preferences in P. nipponicus. In contrast, upper and adaxial surfaces were preferred by B. obovatus. Thus, prey and predatory mites accumulated on different sides of leaves. Presence of the predator also indirectly decreased egg production in B. obovatus. Brevipalpus obovatus females actively avoided leaf surfaces with elevated predator numbers; these females shifted their distributions and changed oviposition sites to leaf surfaces with fewer predators. In consequence, B. obovatus eggs on the upper sides of leaves were less frequently preyed upon than were those on lower sides. We suggest that upper leaf-surface exploitation in this particular herbivorous mite species mitigates predation risk from phytoseiid mites, which prefer lower leaf surfaces.     

Survival, Growth, and Localization of Epiphytic Fitness Mutants of Pseudomonas syringae on Leaves

Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated onto and into plant leaves. For example, while none showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular spaces of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sites protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possibly several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes.     

Behavioral Plasticity in Probing by <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera,Liviidae): Ingestion from Phloem Versus Xylem is Influenced by Leaf Age and Surface

Diaphorina citri is a major pest of citrus because it transmits Candidatus Liberibacter asiaticus, a phloem-limited bacterium that putatively causes Huanglongbing (HLB). The disease moves slowly through a tree, and the vector facilitates further within-tree movement via transmission of the pathogen. However, this only happens when D. citri stylets contact the phloem, to inoculate bacteria during phloem salivation and acquire bacteria during phloem sap ingestion. Behavioral changes in D. citri associated with different plant parts would affect how long it takes to reach phloem and how long the psyllids stays in phloem to ingest, thereby influencing the risk of disease spread. D. citri feeding was recorded on the abaxial and adaxial surfaces of mature and immature citrus leaves. Adults in the field can be found on these surfaces at all times of year. On abaxial surface of immature leaves, phloem salivation would occur after 11 h on average, but rarely as soon as 0.56 h. The corresponding values on mature leaves were 16 and 2.7. In general, psyllids spent more time ingesting phloem sap on immature leaves than on mature leaves. Psyllids on abaxial surfaces spent more time ingesting from phloem, though the strength of this effect was less than for immature versus mature leaves. In contrast, xylem ingestion increased on mature leaves compared with young. The biological differences that could produce this outcome are discussed. The results discussed herein are of relevance to further studies on the efficacy of an insecticide to act quickly enough to prevent pathogen transmission.     

Variability of Bacterial Community Composition on Leaves Between and Within Plant Species

The phyllosphere is one of the largest habitats for terrestrial microorganisms. To gain a better insight into the factors underlying the composition of bacterial communities inhabiting leaf surfaces we performed culture-dependent and independent (Denaturing Gradient Gel Electrophoresis) analyses on the bacteria associated with the leaves of three plant species: Amygdalus communis, Citrus paradisi, and Nicotiana glauca. We found that the culturable classes Bacilli and Actinobacteria were the predominant classes on the phyllosphere of all three plant species. In contrast to this consistency on the bacterial class level, we found a significant variation on the bacterial species-level based on the culturable methods. Although some variation was detected among individual plants within one plant species, the inter-specific variability exceeded the intra-specific variability. C. paradisi leaf surface had the highest predicted total species richness (Chao 2 and ICE) and the highest species diversity (βw) among the three plant species. Our findings demonstrate that environmental conditions, mainly the plant species within a site, govern the bacterial community composition on leaf surfaces.     

Tumor induction by agrobacterium involves attachment of the bacterium to a site on the host plant cell wall

Cell wall preparations from primary bean leaves were found to inhibit tumor initiation by Agrobacterium tumefaciens strain B6 when inoculated with the bacteria on bean leaves. Membrane fractions from these same leaves were noninhibitory. The cell walls were effective when applied prior to or with bacteria, but application of cell walls about 15 minutes after bacteria did not affect the number of tumors initiated. Much of the inhibitory activity of the plant cell walls was eliminated by pretreatment with dead site-attaching bacteria or with lipopolysaccharide from these bacteria. Cells and lipopolysaccharide from non-site-attaching agrobacteria had no effect on the activity of the plant cell walls. About 30% inhibition of tumor initiation was obtained with plant cell walls at 50 μg/ml dry weight, and at 10 mg/ml dry weight about 70% inhibition was typical. Both early and late appearing tumors were affected by the cell walls, indicating that they do not exclusively affect tumors arising from either small or large wounds. These data show that plant cell walls but not membranes contain surfaces to which A. tumefaciens adheres and these exhibit the specificity typical of the host site to which virulent agrobacteria must attach to induce tumors. It is concluded that some portion of wound-exposed plant cell wall constitutes the host adherence site in Agrobacterium infections.     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Water relations of the upper and lower surfaces of maize leaves

The objective of this study was to determine if the two surfaces of a leaf had different, or the same, water potentials. Maize (Zea mays L. cv. Cargill 805) was the test plant. During an 11 day period, the water potentials of the upper and lower surfaces of the maize leaves were measured daily with anin situ thermocouple psychrometer under growth-room conditions. Plants were grown in pots with a well-watered, commercial greenhouse soil. Stomatal resistance also was measured. The water potential of the upper surface was less negative (more wet) than that of the lower surface. The overall average of the water potential of the upper and lower surfaces was ?1.48 and ?2.07 MPa, respectively. The stomatal resistance of the upper surface was greater than that of the lower surface. The average stomatal resistance during the experiment was 780 and 600 s m^?1 for the upper and lower surfaces, respectively. Since the upper and lower surfaces were not at the same water potential, the results indicated that the parallel-resistance equation, used to combine resistances on the two surfaces of a leaf, was not strictly valid when applied to the maize leaves, as the law assumes equal potentials on the two surfaces.     

The geometry of unfolding tree leaves

Leaves of hornbeam (Carpinus betulus) and beech (Fagus sylvaticus) were modelled to a first approximation as plane surfaces, with straight parallel folds, using numerical methods. In both species the lateral veins, when the leaves are outstretched, are angled at 30 to 50 degrees from the centre vein. A higher angle allows the leaf to be folded more compactly within the bud, but it takes longer to expand. This may allow the plant to optimize the timing of leaf deployment with ecological and physiological conditions.     

The foliar micromorphology of Solanum pseudocapsicum

Foliar micromorphology of Solanum pseudocapsicum was investigated by electron microscopical examination. The leaves are characterized by anisocytic stomata which are more abundant on the abaxial surfaces. The leaves have short multicellular glandular trichomes sparsely distributed over the entire leaf surfaces. Crystal deposits were also observed on the surfaces above the stomata. Energy dispersive X-ray spectroscopy-SEM showed that Al, K, Na and Si were the major constituents of the crystals analyzed. The presence of glandular trichomes in this plant could be the source of poisonous compounds that are characteristic of this species.     

Leaf paraheliotropism in Styrax camporum confers increased light use efficiency and advantageous photosynthetic responses rather than photoprotection

Styrax caporum is a native shrub from the Brazilian savanna. Most of its leaves are diaheliotropic, whereas some are paraheliotropic, mainly at noon. A previous study of this species revealed higher stomatal conductance (gs) and transpiration rates (E) in para- compared to diaheliotropic leaves, and a rise in CO₂ assimilation rates (A) with an increase of irradiance for paraheliotropic leaves. We hypothesized that this species exploits the paraheliotropism to enhance the light use efficiency, and that it is detected only if gas exchange is measured with light interception by both leaf surfaces. Gas exchange was measured with devices that enabled light interception on only one of the leaf surfaces and with devices that enabled light interception by both leaf surfaces. Water relations, relative reflected light intensity, leaf temperature (T_l), and leaf anatomical analyses were also performed. When both leaf surfaces were illuminated, a higher A, E, and gs were observed in para- compared to diaheliotropic leaves; however, A did not depend on gs, which did not influence CO₂ accumulation in the stomatal cavity (Ci). When only the adaxial leaf surface was illuminated, a greater A was detected for para- than for diaheliotropic leaves only at 11:00 h; no differences in T_l were observed between leaf types. Light curves revealed that under non-saturating light the adaxial side of paraheliotropic leaves had higher A than the abaxial side, but they showed similar values under saturating light. Although the abaxial leaf side was highly reflective, both surfaces presented the same response pattern for green light reflection, which can be explained by the compact spongy parenchyma observed in the leaves, increasing light use efficiency in terms of CO₂ consumption for paraheliotropic leaves. We propose that paraheliotropism in S. camporum is not related to leaf heat avoidance or photoprotection.     

Competitive Ability and Survival in Soil of Pseudomonas Strain 679-2, a Dominant, Nonobligate Bacterial Predator of Bacteria

A copper-resistant, nonobligate, bacterial predator of bacteria was isolated from soil. It was a Pseudomonas species, designated strain 679-2. It attacked most other nonobligate bacterial predators and hence could control their predatory and other activities in nature. It also inhibited various fungi. It attached to prey cells and produced a toxic, copper-related, growth initiation factor like that produced by Cupriavidus necator. In addition, it produced a second, novel compound that was both antibacterial and antifungal. Strain 679-2 appeared to have only a very limited natural occurrence. It was found only in the soil from one small area in one field. It was absent on the leaves of the plant species that were examined. Regardless of its rarity, however, it was highly competitive in soil. An inoculum consisting of only a few cells added to soil multiplied rapidly to become a major component of the soil microflora within 24 h. A small amount of glutamic acid could be added along with the cells to stimulate production of the toxic compounds noted above, but this was not necessary. After this multiplication, or when large numbers of cells were added to soil, the numbers decreased only slowly during the next several months. Cell survival also was good on plant leaves. The survival in soil and on plant leaves occurred in both laboratory and field experiments. Other than desiccation, the natural mechanism for controlling the numbers or activities of strain 679-2 in soil is not known. The various characteristics of this bacterium, as noted above, are of particular interest because they indicate a possible use of the cells or inhibitor compounds for controlling organisms in soil or on plant surfaces.     

Weevil borne microbes contribute as much to the reduction of photosynthesis in water hyacinth as does herbivory

Arthropods released for weed biocontrol can have effects other than simply removing biomass and frequently decrease photosynthetic rate more than can be attributed to the mere loss of photosynthetic surface area. Some of this effect may result because biological control agents facilitate the transfer and ingress of deleterious microbes into plant tissues on which they feed. We evaluated this facilitation effect using water hyacinth (Eichhornia crassipes) and a weevil (Neochetina eichhorniae) and compared the reductions in photosynthetic rates between leaves subject to herbivory by adult weevils sterilized with 3.5% chlorine bleach, to those that were unsterilized. The results showed that weevils carried both fungi and bacteria, transferred these to leaves on which they fed, and that microbes and biomass removal contributed almost equally to the 37% decrease in photosynthetic productivity. Hence, maximising the effectiveness of using arthropods that damage leaf surfaces for biocontrol requires the presence of microorganisms that are deleterious to plants.