The composition and temporal variation of the phylloplane microflora of Photinia glabra confirms to the general pattern displayed by those of other plant species. Limitations in the cultural methods widely employed in phylloplane studies have been highlighted by the results. The need to employ several cultural techniques in studies of the phylloplane microbiology is demonstrated by the contrasting population estimates provided by these techniques. Advantages of using a range of techniques in quantitative studies of the phylloplane have been elaborated in earlier studies (Lindsey and Pugh, 1976). The leaf print technique appears to provide more credible data, though this method fails to achieve complete isolation of the microflora. Leaf washing technique provided extremely low estimates of the microflora. Incomplete spore removal rather than biased sampling of washings would have caused such low population estimates by this technique. This was confirmed by the residual microflora (which constituted a high proportion of the total microflora) on the washed leaf surfaces and isolated by the leaf print technique. All methods accurately record seasonal trends in fungal population variations, suggesting that any single cultural method could provide reliable data regarding seasonal variations of the phylloplane microflora. However, the suitability of a sole cultural method in studies aimed at more precise quantitative data is questionable. A possible solution in such cases would be to adopt a combination of techniques that complement each other. The leaf washing technique and leaf print technique could be employed in tandem to achieve near complete isolation of the microflora. The summation of the counts provided by the two techniques could be regarded as a more accurate account of the microflora on leaf surfaces.
Scanning Electron Microscope is a useful tool in the verification of actual fungal growth on the leaf surface. It provides information regarding the forms of fungal propagules on leaf surfaces, their spatial distribution and most importantly, their physiological stage. In the present experiment, cultural methods indicated a preponderance of E. nigrum on the leaf surface. An interpretation based on this information would have been erroneous. The picture provided by the SEM, of other fungi actively growing on the leaf surface, was crucial in correctly evaluating the leaf surface as a substrate for microbial growth. Samples can be examined under the SEM while the natural distribution of microorganisms on the leaf surface is preserved. This is achieved by obviating the need for bleaching, essential in the light microscopic examination. However, the sample preparation procedure involved in the SEM can cause collapse of fungal propagules. This peril can be avoided by paying due care. The major drawback of the SEM is its inability to facilitate examination of the
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whole leaf surface. In the absence of this facility, information gained may not appear interconnected. It is also a relatively expensive method in terms of costs involved in sample preparation. Scanning Electron Microscope emerges as a method that could provide details necessary to explain uncertainties regarding fungal activity on leaf surface. However, this does not warrant its routine application in every study of the phylloplane.
Routine isolation of the microflora on to a commonly used synthetic culture medium could demonstrate only a section of the microflora present on leaf surfaces. Using tap water agar, Dickinson (1973) did not record the presence of Aureobasidium pullulans on barley leaves, yet recorded high counts of this yeast when leaf washings were plated on PDA. In addition to such complete exclusions in culture, some organisms favoured by the sources and composition of nutrients in a particular culture medium could overgrow certain others. Differences among the microflora sustained by different culture media in this experiment could be explained on the basis of differences in nutrient composition and carbon sources. CDA and PDA, the media which sustained higher number of colonies are comparable in their contents of major nutrients (Na, K, Mg, Fe) except carbon. The reason for the superiority of PDA over CDA could be the readily available carbon in PDA. Dextrose, a sugar easily absorbed and ingested, could encourage the germination and growth of many of the Fungi Imperfecti. The source of carbon in CDA, sucrose, has to be enzymatically hydrolysed before absorption. Inadequate carbon supplies would have hampered germination and growth of organisms on CDA during the 5 d incubation. Com Meal Agar and OMA obviously do not contain the range of nutrients needed to support the growth of such a diverse group of fungi. Practical implication of this observation is that sampling of leaf surface microflora has to be done on a number of media in order to choose the medium which renders the broadest spectrum of fungi. Yet, many of the reported studies of the phylloplane have been carried out without adequate attention paid to these aspects. Variation among replicates indicate the dependance of leaf surface microflora on extraneous factors which determine the size and composition of the aerial microflora. The populations of E. nigrum and A. alternata, which occurred mainly as spores would have reflected their concentration in the aerial microflora. The fungal propagule content in the atmosphere is determined by the environmental and other factors governing growth and sporulation of fungi elsewhere. The variation in yeast populations, which were found to be actively growing on leaf surfaces, could be attributed to different degrees of isolation achieved in each culture plate and the variation in the proportion of yeasts in reproductive stage. Yeasts are generally the most difficult group of microorganisms to isolate by the leaf
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print method. Substantial numbers of yeast cells have been found to remain on leaf surfaces after ten repeated prints were made on agar (Parberry et al., 1981). Such inconsistencies would have contributed to the high variation in data. The yeasts in reproductive stage are comparatively easily isolated due to the easy detachment of the buds. However, the proportion of yeast cells in reproductive stage may be subject to unstable conditions in the boundary layer.
Species composition of the fungi on leaf surfaces of Photinia glabra is similar to those found on other plant species. The genera frequently isolated from leaf surfaces form the bulk of the air spora (Last, 1955; Collins and Hayes, 1976). The fungal flora is dominated by the three species E. nigrum, C. cladosporioides and A. alternate. Among the yeasts, R. glutinis, S. roseus and C. albidus predominated. The occurrence of fungi mainly in the form of spores (with the exception of C. cladosporioides), suggests that fungal growth on leaf surfaces is restricted. A feature of the microflora recorded on these leaf surfaces is the absence of Aureobasidium pullulans. This yeast is generally regarded as the inidal colonizer of green leaves and has been reported on most leaf surfaces that have been examined. However, Aureobasidium pullulans has been absent on few plant species including Acer platanoides (Breeze and Dix, 1981) and Ilex aquifolium (Mishra and Dickinson, 1981). In a comparative study of the phylloplane microflora of Paspalum dilatatum, Salix babilonica and Eucalyptus stellulata, A. pullulans was not recorded on any of the three leaf surfaces (Lamb and Brown, 1970). Significantly, in all cases where A. pullulans has not been recorded, E. nigrum has been the major inhabitant of the leaf
surface.
Significant differences in the patterns of colonization between the upper and lower surfaces have also been reported by Ruscoe (1971), Wildman and Parkinson (1979) and Mishra and Dickinson (1981). In these studies, though higher numbers of fungal propagules were consistently recorded on the upper surface, a higher degree of spore germination and hyphal growth was observed on the lower surface. Differences between the microenvironments of the upper and lower surfaces explain this phenomenon. Lower leaf surfaces are known to maintain higher levels of relative humidity due to stomatal transpiration. Drying effect of the solar radiation on lower leaf surfaces is reported to be slower due to rougher microtopography. This reduces the direct detrimental effect of drying on microorganisms and, indirectly support microbial growth by prolonging the period during which nutrients could be exuded (Waggoner, 1965). Cultural techniques commonly employed in the study of the phylloplane however, do not provide information
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relating to topographic variation of the microflora. In fact, a study of the phylloplane not accompanied by the direct observation techniques could lead to erroneous conclusions regarding fungi that are active in this environment.
Correlation exhibited by the microbial populations on leaf surfaces with weather parameters provides an insight into the population variations, and explains the unpredictability and variation found in population levels and composition. Previous attempts to explain seasonal variations in leaf surface microbial populations have been speculative. Last (1955) attributed the summer increase of Sporobolomyces to increased leaf age, temperature and air movement. Pennycook and Newhook (1983) considered daily duration and intensity of insolation as causative factors. Hayes (1981, 1982) first attempted to correlate statistically climatic regimes with phylloplane microbial populations, though with negative results. Results presented here are supportive of his suggestion that more remote meteorological events may not be without critical influence. The meteorological factors exhibiting correlation here are the average (temperature and relative humidity) and total (rainfall) values for the fortnight preceding the date of sampling. The effect on E. nigrum could be through the increase in concentration of these spores in the aerial microflora. The correlation exhibited by the total microbial population is due to E. nigrum which constitutes nearly 80% of the population. Lack of correlation with the established fungus C. cladosporioides and the yeasts could be due to the inconsistencies in isolating these populations described above.
These results suggest that the leaf surface as an ecological niche does not harbour a specialized microbial population similar to those of litter, soil and water, but acts as a refuge for numerous airborne fungal propagules. Studies aimed at an accurate image of this environment should employ a variety of methods of observation. Leaf surface microflora appear to be dependant on weather parameters for successful establishment.
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