Phylloplane Microorganisms Research Articles

Phylloplane microorganisms and induction of systemic resistance against pathogen Alternaria solani in tomato (Solanum lycopersicum L.)

Phylloplane microorganisms and induction of systemic resistance against pathogen Alternaria solani in tomato (Solanum lycopersicum L.)

Sampling of Plant Material to Study Endophytes in Small, Large, and Woody Plants.

The study of fungal, bacterial, and other endophytic microorganisms using high throughput DNA sequencing requires sampling and processing of plant material that eliminates phylloplane microorganisms and retains those inside the plant compartment. Leaves, stems, roots, and other plant tissues are removed from the plant, washed, surface sterilized, and stored for downstream applications. Especially in ecological studies, field work for sample collection may take place in remote locations where laboratory equipment and resources are rudimentary, and accessing samples from target plants can be challenging. This chapter serves as a guide to basic protocols in the design and sample collection for studies focused on the endophytes of leaf, stem, and root tissues.

Witches\u2019 broom resistant genotype CCN51 shows greater diversity of symbiont bacteria in its phylloplane than susceptible genotype catongo

BackgroundTheobroma cacao L. (cacao) is a perennial tropical tree, endemic to rainforests of the Amazon Basin. Large populations of bacteria live on leaf surfaces and these phylloplane microorganisms can have important effects on plant health. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated studies of the phylloplane microbiome. In this study, we characterized the bacterial microbiome of the phylloplane of the catongo genotype (susceptible to witch’s broom) and CCN51 (resistant). Bacterial microbiome was determined by sequencing the V3-V4 region of the bacterial 16S rRNA gene.ResultsAfter the pre-processing, a total of 1.7 million reads were considered. In total, 106 genera of bacteria were characterized. Proteobacteria was the predominant phylum in both genotypes. The exclusive genera of Catongo showed activity in the protection against UV radiation and in the transport of substrates. CCN51 presented genus that act in the biological control and inhibition in several taxonomic groups. Genotype CCN51 presented greater diversity of microorganisms in comparison to the Catongo genotype and the total community was different between both. Scanning electron microscopy analysis of leaves revealed that on the phylloplane, many bacterial occur in large aggregates in several regions of the surface and isolated nearby to the stomata.ConclusionsWe describe for the first time the phylloplane bacterial communities of T. cacao. The Genotype CCN51, resistant to the witch’s broom, has a greater diversity of bacterial microbioma in comparison to Catongo and a greater amount of exclusive microorganisms in the phylloplane with antagonistic action against phytopathogens.

Phylloplane bacteria of Jatropha curcas: diversity, metabolic characteristics, and growth-promoting attributes towards vigor of maize seedling.

The complex role of phylloplane microorganisms is less understood than that of rhizospheric microorganisms in lieu of their pivotal role in plant's sustainability. This experiment aims to study the diversity of the culturable phylloplane bacteria of Jatropha curcas and evaluate their growth-promoting activities towards maize seedling vigor. Heterotrophic bacteria were isolated from the phylloplane of J. curcas and their 16S rRNA genes were sequenced. Sequences of the 16S rRNA gene were very similar to those of species belonging to the classes Bacillales (50%), Gammaproteobacteria (21.8%), Betaproteobacteria (15.6%), and Alphaproteobacteria (12.5%). The phylloplane bacteria preferred to utilize alcohol rather than monosaccharides and polysaccharides as a carbon source. Isolates exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase, phosphatase, potassium solubilization, and indole acetic acid (IAA) production activities. The phosphate-solubilizing capacity (mg of PO4 solubilized by 108 cells) varied from 0.04 to 0.21. The IAA production potential (μg IAA produced by 108 cells in 48 h) of the isolates varied from 0.41 to 9.29. Inoculation of the isolates to maize seed significantly increased shoot and root lengths of maize seedlings. A linear regression model of the plant-growth-promoting activities significantly correlated (p < 0.01) with the growth parameters. Similarly, a correspondence analysis categorized ACC deaminase and IAA production as the major factors contributing 41% and 13.8% variation, respectively, to the growth of maize seedlings.

Phylloplane microorganisms as a potential biocontrol agent against Helminthosporium oryzae Breda de Hann, the incitant of rice brown spot

Fifteen pathogenic isolates of Helminthosporium oryzae were established from 15 different rice growing areas representing nine districts: Madurai, Theni, Thanjavur, Thiruvarur, Nagapatinum, Tirunelveli, Trichy, Ariyalur and Cuddalore district of Tamil Nadu. The symptoms initially appeared on the leaves as small, oval, dark brown to black spots. Under favourable conditions, the fungus attacks grain and caused grain discoloration. The isolate, collected from Ammapettai of Thanjavur district was the most virulent (Grade 7.47, PDI 82.96) on rice plants followed by Trichy isolate I2 (Grade 7.26; PDI 80.74) while I15 (Cumbum) was the least virulent (Grade 1.8, PDI 20.00). The isolates of H. oryzae varied in size (length and width) of the conidia, colour and the number of cells per conidium. The maximum (39.1 μm) length of the conidium was observed in I10 followed by I11 (37.3 μm). The maximum (17.1 μm) conidial width was observed in I8. The colour of the conidium varied from light brown (isolates I3, I6 and I14) to brown, while there was no appreciable difference in the conidial shape. The number of cells varied from 2 – 4 among the isolates. Six phylloplane microorganisms were tested against the virulent isolate of H. oryzae. Among them, Cladasporium spp was very effective in inhibiting the mycelial growth and spore germination of H. oryzae followed by Penicillum spp and Aspergillus flavus while Bacillus subtilis was least effective in inhibiting the mycelial growth and spore germination of H. oryzae.

Influence of roadside pollution on the phylloplane microbial community of Alnus nepalensis (Betulaceae)

The North Eastern region of India is undergoing industrial development at a faster rate than expected. Roads form the main system of transportation and communication owing to the hilly topography of the region. Automobiles discharge a number of gaseous and trace metal contaminants. Human activities like stone grinding, road construction and sand milling also increase the atmospheric dust and heavy metal contaminant level. These contaminants get settled on leaf surfaces at roadsides and enter in contact with phylloplane microorganisms. This study compares microorganisms on leaf surfaces of alder (Alnus nepalensis (Betulaceae)) on roadside and non-roadside environments. Two sites dominated by alder were selected. One at a busy road intersection on the National Highway no. 44 in Shillong with high traffic density (8 000-9 000 heavy vehicles/day), taken as the polluted site and the other one in a forest approximately 500 m away from the roadside considered as the unpolluted site. Analysis of phylloplane microorganisms, lead, zinc, copper, cadmium and sulphur was carried out from leaves. The bacterial population was higher at the unpolluted site. Bacterial population showed a significant negative correlation with lead, zinc, copper, cadmium and sulphur. Similarly, fungal population was higher at the unpolluted site. A total of 29 fungal species were isolated from the phylloplane of A. nepalensis (polluted site 16 species; unpolluted site 28 species). Some fungal forms like Mortierella sp., Fusarium oxysporum and Aureobasidium pollulans were dominant in the polluted site. Numbers of phylloplane fungi and bacteria were significantly reduced in the polluted site. The correlation coefficient indicated a detrimental effect of metals like lead, zinc, copper, cadmium and sulphur on the microbial community of leaf surfaces. The specificity of certain fungi to the unpolluted site may be attributed to their sensitivity to pollution. The predominance of Aureobasidium pollulans, Fusarium oxysporum and Mortierella sp. in the polluted site may be due to their resistance to pollutants in roadside conditions.

Evaluation of phylloplane microorganisms for biological control of camellia flower blight

Microorganisms isolated from camellia blooms were evaluated for their ability to protect camellia flowers against ascospore infection by Ciborinia camelliae. of the 277 isolates tested in camellia petal assays, Bacillus sp. LU1006, Bacillus subtilis LU1007, Pseudomonas sp. A3/B1, Pseudomonas marginalis LU1009, Pseudomonasfluorescens LU1004, Aureobasidium sp. Ch3F3, Cladosporium sp. LU172 and Cladosporium cladosporioides LU174 provided almost total protection against infection. However, when these isolates were suspended in water alone, or with either an organo-silicon surfactant (0.01% Break-Thru) or an ultraviolet protectant sticker-oil (Codacide), and sprayed onto 5-year-old potted Camellia japonica plants, they failed to reduce the incidence of camellia flower blight. Although these microorganisms protected individually sprayed petals from ascospore infection, they were unable to colonise petals on flowers sufficiently well to provide protection against camellia flower blight in field conditions.

Degradation and Detoxification of the Dihydrochalcone Phloridzin by &lt;I&gt;Enterobacter agglomerans&lt;/I&gt;, a Bacterium Associated with the Apple Pest, &lt;I&gt;Rhagoletis pomonella&lt;/I&gt; (Walsh) (Diptera: Tephritidae)

Phloridzin, a plant-derived compound toxic to Rhagoletis pomonella (Walsh), the apple maggot fly, was degraded and detoxified by the bacterium Enterobacter agglomerans. Spectrophotometric and thin-layer chromatography data showed that E. agglomerans begins to use the glycoside in aqueous solution after 6 h of incubation at 24°C. All apple maggot flies that fed on three different concentrations of sterilized phloridzin solutions died within 24 h. Incubation of E. agglomerans in 0.001 and 0.01 M aqueous phloridzin for 3 d eliminated toxicity for apple maggot flies but most died after being fed a 1 M solution. Plant leachate containing phenolic compounds extracted from McIntosh apple leaves also was used by E. agglomerans. Enterobacter agglomerans entered logarithmic growth more quickly in leachate samples than in solutions of commercially-purified phloridzin. Protein, amino acid, and reducing sugar content of the leachate increased after incubation with E. agglomerans for 12 h. Reducing sugar content decreased, however, after 24 h of incubation, whereas protein and amino acid content continued to increase. Scanning electron micrographs of leachate intact on apple leaf surfaces revealed the presence of healthy rod-shaped bacteria and yeast aggregating about it. These images suggest that phylloplane microorganisms use leachate as a source of nutrients and this activity may affect the potency of plant defensive chemicals. The potential benefits afforded by the catabolic activities of phylloplane and/or gut microorganisms on plant compounds to apple maggot flies are discussed.

Single-leaf resolution of the temporal population dynamics of Aureobasidium pullulans on apple leaves.

The abundance of phylloplane microorganisms typically varies over several orders of magnitude among leaves sampled concurrently. Because the methods traditionally used to sample leaves are destructive, it has remained unclear whether this high variability is due to fixed differences in habitat quality among leaves or to asynchronous temporal variation in the microbial population density on individual leaves. We developed a novel semidestructive assay to repeatedly sample the same apple leaves from orchard trees over time by removing progressively more proximal approximately 1-cm-wide transverse segments. Aureobasidium pullulans densities were determined by standard leaf homogenization and plating procedures and were expressed as CFU per square centimeter of segment. The A. pullulans population densities among leaves were lognormally distributed. The variability in A. pullulans population densities among subsections of a given leaf was one-third to one-ninth the variability among whole leaves harvested concurrently. Sequential harvesting of leaf segments did not result in detectable changes in A. pullulans density on residual leaf surfaces. These findings implied that we could infer whole-leaf A. pullulans densities over time by using partial leaves. When this successive sampling regimen was applied over the course of multiple 7- to 8-day experiments, the among-leaf effects were virtually always the predominant source of variance in A. pullulans density estimates. Changes in A. pullulans density tended to be synchronous among leaves, such that the rank order of leaves arrayed with respect to A. pullulans density was largely maintained through time. Occasional periods of asynchrony were observed, but idiosyncratic changes in A. pullulans density did not contribute appreciably to variation in the distribution of populations among leaves. This suggests that persistent differences in habitat (leaf) quality are primarily responsible for the variation in A. pullulans density among leaves in nature.

UV‐B Biodosimetry in Turfgrass Canopies

Phylloplane microorganisms are affected by ultraviolet (UV) radiation penetrating into plant canopies, but data as to the relationships between microorganism activity and canopy UV levels is lacking. Current instrumentation and modeling systems are inadequate to analyze canopy radiation environments at scales relevant to microorganisms. A biological dosimeter system was developed for measuring UV‐B in turfgrass and other compact canopies. Cell suspensions of a DNA repair‐deficient strain of Escherichia coli (CSRO6) were enclosed in packets (0.2‐mL volume) of UV‐transmissible polyethylene. After the packets were exposed to sunlight, numbers of surviving bacteria were determined. The log percent survival was found to be linearly related to accumulative UV‐B dosage, as measured with a broad‐band UV‐B radiometer, but was not related to UV‐A dosage. In one experiment, the performance of the biodosimeter system was compared with that of a miniature UV‐B radiometer mounted in soil‐level tracks in eight plots of tall fescue (Festuca arundinacea Schreb.) that varied in leaf area index (LAI). The two methods yielded similar mean transmittance values that decreased with increasing LAI, closely fitting Beer's law. A similar relationship was found in a second experiment, in which biodosimeter packets were placed at the base of undisturbed tall fescue canopies. The packets also revealed considerable variation in transmittance possibly because of localized shading and sun flecks in the natural canopies. In a third experiment, direct and diffuse UV‐B at different heights within a tall fescue canopy was measured by packets attached to narrow, flat wooden sticks simulating grass leaves. This method has potential as a tool to capture the variability in UV levels related to nonuniformity in canopy structure, depth in a canopy, and leaf orientation.

UV-B Biodosimetry in Turfgrass Canopies

Phylloplane microorganisms are affected by ultraviolet (UV) radiation penetrating into plant canopies, but data as to the relationships between microorganism activity and canopy UV levels is lacking. Current instrumentation and modeling systems are inadequate to analyze canopy radiation environments at scales relevant to microorganisms. A biological dosimeter system was developed for measuring UV-B in turfgrass and other compact canopies. Cell suspensions of a DNA repair-deficient strain of Escherichia coli (CSRO6) were enclosed in packets (0.2-mL volume) of UV-transmissible polyethylene. After the packets were exposed to sunlight, numbers of surviving bacteria were determined. The log percent survival was found to be linearly related to accumulative UV-B dosage, as measured with a broad-band UV-B radiometer, but was not related to UV-A dosage. In one experiment, the performance of the biodosimeter system was compared with that of a miniature UV-B radiometer mounted in soil-level tracks in eight plots of tall fescue (Festuca arundinacea Schreb.) that varied in leaf area index (LAI). The two methods yielded similar mean transmittance values that decreased with increasing LAI, closely fitting Beer's law. A similar relationship was found in a second experiment, in which biodosimeter packets were placed at the base of undisturbed tall fescue canopies. The packets also revealed considerable variation in transmittance possibly because of localized shading and sun flecks in the natural canopies. In a third experiment, direct and diffuse UV-B at different heights within a tall fescue canopy was measured by packets attached to narrow, flat wooden sticks simulating grass leaves. This method has potential as a tool to capture the variability in UV levels related to nonuniformity in canopy structure, depth in a canopy, and leaf orientation.

A novel terrestrial halophilic environment: The phylloplane of Atriplex halimus, a salt-excreting plant

This paper describes the microbial ecosystem found on the leaves of Atriplex halimus, a salt-excreting plant in the central Negev highlands of Israel. Because of the regular nightly occurence of dew at this location, these leaves undergo a diurnal wetting so that phylloplane microorganisms experience large fluctuations in salinity and water activity, as well as tolerate repeated desiccation. During the dry season, in the late spring and summer, a significant amount of salts and organic material coats the leaf surface. During dew events the salt concentration at the leaf surface was calculated to be > 0.4 M. Direct counts of the respiring bacteria on the leaf surface ranged from 1.06×104 to 5.06×105 per cm2. Using a variety of media it was shown that there was limited bacterial diversity which could be cultured, with greater than 90% of the isolates being orange colored Gram-negative rods. Viable counts ranged from 0.32 to 2.32×104 bacteria per cm2 of A. halimus leaf surface. No bacteria capable of nucleating ice were recovered in these studies. The dominant orange pigmented bacterium, identified as a halotolerant Pseudomonas sp., grew optimally at 30°C and at 5% NaCl and was capable of growth in media containing up to 20% NaCl. This bacterium could grow on a variety of organic compounds, including some associated with plant materials. The leaf bacteria were desiccation-tolerant when on the leaf surface or when directly washed off the leaves, but much less so when in isolatd culture. A major component of the tolerance to desiccation is probably related to the compounds on the leaf surface.

A novel terrestrial halophilic environment: The phylloplane of Atriplex halimus, a salt-excreting plant

This paper describes the microbial ecosystem found on the leaves of Atriplex halimus, a salt-excreting plant in the central Negev highlands of Israel. Because of the regular nightly occurence of dew at this location, these leaves undergo a diurnal wetting so that phylloplane microorganisms experience large fluctuations in salinity and water activity, as well as tolerate repeated desiccation. During the dry season, in the late spring and summer, a significant amount of salts and organic material coats the leaf surface. During dew events the salt concentration at the leaf surface was calculated to be > 0.4 M. Direct counts of the respiring bacteria on the leaf surface ranged from 1.06×104 to 5.06×105 per cm2. Using a variety of media it was shown that there was limited bacterial diversity which could be cultured, with greater than 90% of the isolates being orange colored Gram-negative rods. Viable counts ranged from 0.32 to 2.32×104 bacteria per cm2 of A. halimus leaf surface. No bacteria capable of nucleating ice were recovered in these studies. The dominant orange pigmented bacterium, identified as a halotolerant Pseudomonas sp., grew optimally at 30°C and at 5% NaCl and was capable of growth in media containing up to 20% NaCl. This bacterium could grow on a variety of organic compounds, including some associated with plant materials. The leaf bacteria were desiccation-tolerant when on the leaf surface or when directly washed off the leaves, but much less so when in isolatd culture. A major component of the tolerance to desiccation is probably related to the compounds on the leaf surface.

An artificial wax substrate for the in vitro study of phylloplane micro-organisms

An artificial substrate consisting of a wax-based medium amended with nutrients has been developed for the in vitro study of microbial interactions on the phylloplane. Inoculation of bacteria and yeasts onto the surface demonstrated that these micro-organisms remain viable for up to 13 days (e.g. Aureobasidium pullulans and proliferate on the surface under conditions of low humidity. The growth rate was slower (doubling time 1.2 days) than on nutrient agar or in nutrient broth. The artificial leaf wax substrate supported a lower bacterial population (10 6–10 7 c.f.u. plate −1) than conventional nutrient substrates. Inoculation with Aureobasidium pullulans in high (10 9 c.f.u. plate −1) or low (10 4 c.f.u. plate −1) numbers indicated that the surface could support between 16 × 10 4–16 × 10 5 c.f.u. per cm 2. In the absence of incorporated nutrients, yeast numbers were found not to increase. Spore germination and mycelial elongation of filamentous fungi also occurred on the artificial leaf wax substrate but rates were reduced in comparison with those on nutrient agar. Inoculation of the artificial leaf substrate with a mixed population of a soil yeast and a phyllosphere-specific yeast resulted in competition between species such that the phyllosphere yeast became dominant.

Enhancement of disease caused by Colletotrichum truncatum in Sesbania exaltata by coinoculating with epiphytic bacteria

Colletotrichum truncatum isolate NRRL 13737 (ARS patent culture No. 18434) is being evaluated for development as a mycoherbicide against the problematic weed Sesbania exaltata. Studies were conducted to determine whether selected phylloplane microorganisms, used as coinoculants, could increase the severity of disease incited by C. truncatum in S. exaltata. Hemp Sesbania seedlings were grown in a variety of soils and environments, sprayed with conidia of C. truncatum, and the formation of appressoria was examined on leaves using epifluorescence microscopy. From hemp sesbania plants that supported high levels of appressoria formation, over 200 phyllosphere microorganisms were isolated. Fifteen of 73 microbial isolates assayed stimulated appressoria formation in vitro on cellophane membranes. Five of 8 superior isolates from the in vitro assay also enhanced disease symptoms induced by C. truncatum on S. exaltata compared to seedlings treated with conidia only. Populations of three selected superior isolates remained high on leaves during plant exposure to dew. The superior isolates initiated no apparent symptoms and rarely decreased seedling growth parameters in the absence of C. truncatum. This is the first instance of utilizing phylloplane microorganisms to increase the level of disease incited by a mycoherbicide agent. Microbial facilitators may provide a means of improving the weed control efficacy of mycoherbicides.

Structural changes and associated microbial activity accompanying decomposition of mangrove leaves in Mgeni Estuary

Leaves of Avicennia marina (Forssk.) Vierh. and Bruguiera gymnorrhiza (L.) Lam. were placed in litter bags and allowed to decompose in situ before collection at 7, 14, 21, 42, 64 and 85 days. Material from each collection was examined for structural changes using light microscopy and SEM. Samples of each collection were also cultured for fungi and bacteria. Examination of senescent leaves still attached to the trees revealed the presence of phylloplane micro-organisms. Decomposition in both species followed a similar pattern although the leaves of A. marina decomposed more rapidly than those of B. gymnorrhiza. By day 7 there were no obvious changes in leaf structure, initial decomposition of the leaf base was the only apparent change in structure on day 14; by day 21 portions of the cuticular layer had separated from the leaf tissue; on day 42 most of the leaves were split longitudinally, separating the adaxial and abaxial surfaces; in the day-63 material, fragmentation of the leaves was evident; further fragmentation and ‘skeletonization’ of leaves was observed after 85 days of decomposition. Both bacteria and fungi were important in the decomposition process. Leaves of both mangroves were found to harbour 20–25 species of fungi. This work appeared to confirm the significance of micro-organisms in the estuarine food web.

Nitrogenase activity associated with leaf, root, and rhizosphere soils in an Indian tropical forest

N2 fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N2-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.

Phylloplane micro-organisms of Rosa cv. Picadilly following infection by Diplocarpon rosae

The phylloplane microflora of the hybrid tea rose cultivar Picadilly was studied for two growing seasons on healthy leaves and on leaves infected by Diplocarpon rosae . Large increases in numbers of yeasts and bacteria on healthy leaves were noted from the end of July until the beginning of October. Late in the growing season numbers of all types of microbes increased dramatically. Following infection by D. rosae numbers of microbes generally showed a marked increase, but this was not always sustained. Cryptococcus laurentii and Micrococcus sp. populations on infected leaves were 3–4 times those on correspondingly healthy leaves. The species composition and changes in numbers of phylloplane microbes are compared with descriptions of microfloras isolated from leaves of other plants and possible reasons for the observed differences are discussed.

FUNGAL COLONIZATION OF PISUM LEAVES

Systematic studies on phylloplane microorganisms are required to answer questions concerning pathogenic and saprophytic destruction of leaves. Pisum leaves, which are excellent experimental material, have been examined during the entire growing season of the plants, and beyond, in the first stages of decomposition. Green leaves were not extensively colonized by filamentous fungi. At the onset of senescence, a dramatic increase was observed in the activities of several saprophytes and on newly dead leaves colonization proceeded even more quickly. Cladosporium, Stemphylium, and Alternaria were frequently observed in the first wave of saprophytes. The first two genera sporulated soon after the leaves died. The increase in saprophyte activity was considered to be primarily a result of alterations in leaf physiology though the possibly beneficial influence of changes in various climatic factors may not be neglected.