Sri Lankan J. Agric. Sci. Vol.

42 - 2005, 94 - 104

MICROBIAL POPULATION DYNAMICS OF THE PHYLLOSPHERE OF TEA (CAMELLIA SINENSIS) AND ITS INFLUENCE ON EXOBASIDIUM VEXANS, THE BLISTER BLIGHT PATHOGEN D.M. De Costa and R.M.S.P. Rathnayake Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Sri Lanka and A. Balasuriya Tea Research Institute, St. Coombs, Talawakele, Sri Lanka.

SUMMARY The objective of the present study was to determine the microbial population dynamics of the phyllosphere of tea together with potential antagonistic isolates of bacteria and yeasts against Exobasidium vexans, the blister blight pathogen. Culturable microorganisms dwelling on tea leaves were isolated from mature and immature leaves of seedling and clonal tea in two isolation periods. Potential antagonists of E. vexans were also screened from bacterial and yeast isolates dwelling on the phyllosphere of Camillia japonica, a taxonomically related species of tea and four shade trees grown most frequently in tea estates. Density and diversity of epiphytic microorganisms varied significantly by the maturity level and isolation period but not by the type of tea used. Bacterial and yeast isolates obtained from the phyllosphere of tea did not inhibit spore germination or germtube elongation in vitro when 3 different concentrations (i.e. 5%, 10% and 15% (v/v)) of their cell free extracts were used. Instead, E. vexans spores germinated and showed germtube elongation with the extracts of all the tested bacteria and yeast isolates. No antagonistic isolate was found from the bacterial and yeast isolates obtained from the phyllosphere of Camellia japonica and shade trees.

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INTRODUCTION Tea (Camellia sinensis) is the major plantation crop grown in Sri Lanka and plays a key role in the economy by contributing to one third of the export earnings of the country. Yield of tea is affected by leaf and root diseases and among them blister blight caused by Exobasidium vexans Massee is the most serious leaf disease in Sri Lanka (Arulpragasam et al.,1986). Depending on the prevailing weather conditions, yield losses upto 42% - 50% have been reported due to blister blight (Ordish, 1952). Although gradations in susceptibility have been found among all the known clones of tea (Balasuriya and Kalaichelvan, 2000), tea clones resistant to blister blight are not available. Therefore, blister blight is controlled mainly by application of protectant fungicides, systemic fungicides and antibiotic solutions in combination with protectant fungicides (TRI Advisory Circular, 2002). Blister blight is the only disease for which fungicides are being used extensively over massive land areas on a regular basis and could result in higher production costs and environmental and health risks. Frequent applications of fungicides may leave residues in the final product, sometimes exceeding the acceptable maximum residue limits (1-2 ppm for systemic fungicides) of the international health standards (Balasuriya and Kalaichelvan, 2000). Therefore, integrated disease management with an effective biological control component is highly appropriate in minimizing the usage of pesticides. Phyllosphere is the natural habitat on the leaf surface, which supports the growth of a heterogeneous population of microorganisms comprising of both pathogens and non pathogens. The epiphytic microflora of the phyllosphere accommodate a wide range of organisms including fungi, bacteria, yeast, algae and actinomycetae and could be beneficial or detrimental to the host plant (Andrews,1992; Beattie and Lindow, 1995; Fokkema, 1981). Bacteria, fungi and yeast epiphytes have shown antagonistic effects on a wide range of plant pathogens (Fravel et al., 1998) and attempts have been made to identify antagonistic fungi dwelling on the phyllosphere of tea (Balasuriya and Kalaichelvan, 2000). Epiphytic bacteria and yeast are tolerant to adverse environmental conditions exposed by the phyllosphere (Beattie and Lindow, 1995) and could be used effectively if potential antagonists are found against E. vexans. Since flush of tea is the final product of tea, epiphytic bacteria and yeast would be more appropriate organisms for biological control than fungal epiphytes in maintaining its quality parameters. The present study was conducted to determine population dynamics of phyllosphere epiphytic microorganisms of clonal and seedling tea and its variations with time and leaf maturity. Moreover, the possible antagonistic influence of bacterial and yeast isolates dwelling on the phyllosphere of tea, shade trees and Camellia 95

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japonica on Exobasidium vexans, the blister bight pathogen was determined with the long term objective of using the indigenous microflora in an integrated control of blister blight.

MATERIALS AND METHODS Sources of leaf materials: Epiphytic microorganisms were isolated from mature and tender leaves of seedling and clonal tea collected from St. Coombs estate, Talawakele. Leaf material was obtained from four bushes of seedling tea and a series of clones, which included blister blight resistant (i.e. TRI 2025, TRI 2043 and DT 1) and susceptible (i.e. TRI 2023 and TRI 2024) clones. Leaves from top to the second flush leaf were considered as tender leaves while the rest were considered as mature leaves. Isolations were done during two periods, one from October 2002 to mid November 2002 and another from mid November to end of December 2002. Both mature and immature leaf samples were also collected from Camellia japonica (tea rose) grown for ornamental purposes and shade trees grown at the St. Coombs estate, namely Gravellia robusta, Eucalyptus grandis, Erythrina lithosperma and Cassia spectabilis. Isolations from C. japonica and shade trees were done at the second period from mid November to end of December 2002. Extraction of culturable epiphytic microorganisms: Twenty five leaf disks, 1 cm in diameter each were excised from five leaves collected from a single tree. The disks were vortexed at 1400 rpm for 15 minutes, in 25 ml sterilized water with 0.05% Tween 80. In the case of shade trees with narrow leaf blades, the whole leaf was used for shaking instead of disks. The original leaf washing was diluted and plated on Nutrient Agar (NA) medium according to the dilution plate technique (Cappuccino and Sherman, 1983). The plates were incubated at 25oC for 3 days. The number of colony forming units (cfu) were counted and the total number of each type of colony was estimated based on the dilution factor and the volumes used (Cappuccino and Sherman, 1983). Morphologically distinct bacterial and yeast colonies were purified and maintained on NA. In vitro screening for antagonism: A 1 cm diameter agar disk full of bacteria or yeast was introduced to 50 ml of nutrient broth and incubated in a shaker water bath at 25oC for several days. The cell free extracts were taken by filtering the culture through 0.22 m Millipore filters. Filtered extracts were amended with water agar medium to have a final concentration of 5% (v/v), 10% (v/v) and 15% (v/v) upon sterilization of the water agar medium. Petri plates containing water agar and different concentrations of filter extracts were exposed to twigs of tea having freshly formed blisters under bell-jars, allowing the free fall of E. vexans 96

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spores on to the agar plates over a period of 12 hours. Percentage spore germination and germ tube extension of E. vexans were measured under 10 x 40 magnification after 24 hours of incubation. Control treatments containing nutrient broth with no antagonists in water agar were maintained. Each treatment was replicated three times and 20 spores were randomly measured from each replicate. Variations of microbial population of the phyllosphere were determined by ANOVA and mean separation was done using the least significant difference (LSD). RESULTS Tables 1 and 2 show culturable epiphytic microbial population densities (cfu/ cm2) on mature and immature tea leaves for the first and second isolation periods respectively. Densities of bacterial, fungal and yeast populations existing on the tea phyllosphere and the total phyllosphere epiphytic microbial population differed significantly between the different clonal and seedling teas and between the maturity levels of the tea leaves (p<0.0001). Clonal tea tended to harbour higher total microbial densities than the seedling tea especially in mature leaves. The second isolation period resulted in higher population densities in comparison to the first isolation period. Results clearly showed that bacteria were the prominent type of microorganisms on tea phyllosphere as compared to fungal and yeast epiphytes irrespective of the maturity stage of the leaves and the isolation period. Table 1. Epiphytic Microbial Population Density (cfu/cm2) on Mature and Immature Leaves of Tea Isolated during the First Isolation Period
Clone/Bush TRI 2023 TRI 2024 TRI 2025 TRI 2043 DT 1 Seedling 1 Seedling 2 Seedling 3 Seedling 4 Bacteria 19850a 19600a 15780b 18580a 12470
c

Mature leaves Fungi Yeast 1520b 1520b 2290a 2500a 760

b

Total 21870a 21880a 19590a 21840a

b

Bacteria 15780a,b 14500b,c 10430c 17300a,b 16540

a,b

Immature leaves Fungi Yeast 1270b 760c 2290a 1010b 2030 760c 760c 0d 0d
a,b

Total 17300a,b 16020a,b 13480c 19070a 18820a,b 20350a 17550a,b 15770b 17050a,b

500b 760a 1520a 760b 1010 0c 1270a 250b,c

a,b

250c 0c 760a 760a 250 0d 500c 1010a 250b

c

14240

16290b 15520b 18580a 17000a,b

1520b 1010b 760b 1200b

1010a,b

18820a,b 16530b 20610a 18450a,b

18830a 16290a,b 14760b 16800a,b

Note: Within a given column, means with the same letter are not significantly different at p = 0.05.

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Table 2. Epiphytic Microbial Population Density (cfu/cm2) in Mature and Immature Leaves of Tea Isolated during the Second Isolation Period
Clone/ Bush TRI 2023 TRI 2024 TRI 2025 TRI 2043 DT 1 Seedling 1 Seedling 2 Seedling 3 Seedling 4 Bacteria 36000a 20100b 20870b 28000a,b 23410b 16290b,c 23410b 12980c 21380b Mature leaves Fungi Yeast 1270a,b 3800a 1520a,b 2240a 1270a,b 1010a,b 1270a,b 0b 1270a,b 1010a,b 0c 0c 1520a 250b 250b 0c 0c 0c Total 38280a 23900b 22390b 31760a 24930b 17550b,c 24680b 12980c 22650b Bacteria 15000b 14760b 19600a,b 9670b,c 5600c 22400a 8140b,c 5600c 4830c Immature leaves Fungi Yeast 500b 500b 2520a 0c 760b 1010a,b 1540a,b 0c 760b 760a 760a 0b 250a,b 0b 0b 0b 0b 760a Total 16260a,b 16020a,b 22120a 9920b 6360c 23410a 9680b 5600c 6350c

Note: Within a given column, means with the same letter are not significantly different at p = 0.05.

Mature and immature tea leaves resulted in nine morphologically different bacterial and yeast isolates (i.e. six Gram negative type bacteria, one Gram positive type bacterium and 2 yeast isolates) in the two isolation periods. Higher diversity (i.e. morphologically different colonies) of bacterial and fungal epiphytes was shown in the first isolation period when compared to the second isolation period (Figs 1 and 2). In both isolations, mature leaves harboured higher diversities of fungi and bacteria than the immature leaves. During the isolations, a higher number of morphologically different bacterial colonies were observed in the phyllosphere of clonal tea in comparison to seedling tea.

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Isolation period I
No. of different colony types

7 6 5 4 3 2 1 0

a b a a

Bacteria

Fungi
Types of microorganisms

Yeast

Mature

Immature

Fig 1. Epiphytic Microbial Population Diversity of Mature and Immature Leaves of Clonal and Seedling Tea Isolated at the First Isolation Period. Within each group of microbes, bars with the similar letters are not significantly different at p = 0.05

Isolation period II
No. of different colony types

5 4 3 2 1 0

a b

a b a b

Bacteria

Fungi
Types of microorganisms

Yeast

Mature

Immature

Fig 2. Epiphytic Microbial Population Diversity of Immature Leaves Isolated at the Second Isolation Period. Within each group of microbes, bars with the similar letters are not significantly different at p = 0.05 99

Microbial Population Dynamics of the Phyllosphere of Tea and Its Influence on Exobasidium Vexans, the Blister Blight Pathogen

A higher density of bacteria and fungi were obtained from mature leaves of C. japonica than from its immature leaves (data not shown). C. japonica yielded in three morphologically different bacterial isolates during the two isolation periods, including two Gram positive type and one Gram negative type. Diversity of bacteria, fungi and yeast were higher in mature leaves in comparison to immature leaves of C. japonica. Bacteria, followed by fungi, were the predominant type of epiphytic microorganisms on all the shade trees (Fig 3). Yeast isolates were not found from Erythrina and Eucalyptus, and it was the lowest in Gravellia and Cassia. Three morphologically different Gram positive type bacterial isolates and two Gram negative type isolates were observed in from the phyllosphere of shade trees.

8000 7000 6000 cfu/cm 2 5000 4000 3000 2000 1000 0

a a a,b b c c a b Erythrina Gravellia Casia a b Eucalyptus b c

Shade trees Bacteria Fungi yeast

Fig 3. Epiphytic Microbial Population Density (cfu/cm2) of the Phyllosphere of Four Different Shade Trees Grown in Tea Estates. Within each group of microbes, bars with the similar letters are not significantly different at p = 0.05.

Seventeen morphologically different bacterial and yeast isolates obtained from tea leaves, Camellia japonica and shade trees were tested in vitro for antagonism against E. vexans. Among the 17 epiphytic isolates there were 11 Gram positive bacteria, four Gram negative bacteria and two yeast isolates. Dual culture method (Korsten et al., 1995), which is generally used to observe in vitro antagonism could not be performed for E. vexans as the fungi did not grow on artificial media.

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Therefore, incorporation of filtered bacterial and yeast cell-free extracts into the growth medium was done and spore germination and germ tube elongation were considered as factors to determine the antagonism. All the isolates of bacteria and yeast did not show inhibition or reduction of spore germination or elongation of germtube in comparison to the control treatment at all the tested concentrations.

DISCUSSION Bacteria were the predominant type of microorganisms found in all isolations irrespective of the host species, maturity of leaves and the time of isolation. Density and diversity of epiphytic microbial population were always higher in mature leaves of the tested host species. For favourable microbial colonization on the phyllosphere, a good carbon source for the growth and energy, a nitrogen source and certain essential inorganic molecules must be present (Lindow and Mercier, 2000). Exogenous nutrient sources, such as aphid honey-dew and pollen have been associated with a dramatic increase in the microbial carrying capacities of some leaves (Andrews, 1992). The abundance of such nutrients could vary with plant species, age and growing condition (Andrews, 1992). Also freely available water on the leaf surface and high relative humidity normally encourages the microbial colonization on the phylloplane (Lindow and Mercier, 2000). Therefore, mature leaves have a higher potential to be rich with such favourable factors for microbial nutrition and thereby support higher microbial populations when compared to the immature leaves. Gunasekara (1994) has observed variation in the size of epiphytic microbial populations on tea leaves in different seasons. Slight variations of the epiphytic populations of immature leaves in the two different isolation times could be due to such fluctuations of the environmental factors. The time period in which the second isolation was done was relatively wet due to frequent rains and water was freely available on leaf surfaces. This has apparently supported the presence of higher numbers in microbial populations during the wet season compared to the comparatively drier isolation period 1. Under dry conditions, considerably larger temperature rises have been reported on leaf surfaces (Merrall, 1981).

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The results showed that the density of epiphytic microorganisms has no direct relationship with the diversity of the microorganisms. Despite the higher microbial densities, a comparatively lower number of morphologically-different bacterial and yeast isolates were found from the phyllosphere of the tested host species. Survival and colonization of epiphytic microflora are heavily influenced by the environmental factors to which the phyllosphere is exposed to. As reported by Beattie and Lindow (1995), intense solar radiation, strong winds and drastic fluctuations of day and night temperatures could be the factors that determine the number of dominant bacterial and yeast isolates on the phyllosphere. The density of microflora associated with the phyllosphere of C. japonica was lower than that of C. sinensis. Leaf architecture has a direct influence on microbial colonization. Leaf hairs, trichomes, waxiness of the lamina are some factors that govern the carrying capacity of epiphytes on the phyllosphere (Andrews, 1992). Visual differences such as the more shiny nature and more waxy nature of the leaf lamina were observed with C. japonica as compared to C. sinensis. Though the two plant species belong to the same genus, they may have significant differences in leaf architecture and it could be the reason for variations in microbial harbouring capacities. In the present study, all the bacterial and yeast isolates obtained from the phyllospheres of the host plants showed no inhibitory action on spore germination and germtube elongation. Due to the difficulty of in vitro culture of E. vexans, it was not possible to observe any effect on the radial growth of the colony as is normally done in the dual culture method (Korsten et al., 1995). Also this difficulty in in vitro culturing limited the investigations only to the antagonistic ability through chemical effects. It was not possible to observe whether other modes of antagonism such as competition or parasitism were present. Therefore, the parameters that could be considered were limited to spore germination and germtube elongation. Conducive environments for the germination of conidia are created by plant exudates and epiphytic bacteria that live on plant surfaces. Conidia germination and appresoria formation of Colletotrichum musae and Glomerella cingulata, have been stimulated by specialized chelating agents such as siderophores produced by phyllosphere epiphytic bacteria (Swinburne, 1981). Accordingly, changes in the chemical environment created by the tea phyllosphere epiphytic bacteria could have effects on conidia germination of E. vexans. Hence these aspects need further investigations. Bacteria and yeast are more tolerant than fungi to fluctuating environmental conditions experienced by the phyllosphere (Beattie and Lindow, 1995). Therefore, it was decided to screen natural antagonists of bacteria and yeast 102

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against E. vexans. Culturable epiphytes of bacteria and yeast were considered with a view of developing them in mass scale as a biopesticide of E. vexans. However, no potential antagonistic bacterial or yeast isolates were found from the phyllosphere of tea, Camellia japonica and the shade trees. Therefore, the effectiveness of any other means of biological control of blister blight of tea has to be investigated.

CONCLUSION Density and diversity of epiphytic microorganisms varied significantly by the maturity level and isolation period but not by the type of tea used. Bacterial and yeast isolates obtained from the phyllosphere of C. sinensis, C. japonica, Gravellia robusta, Eucalyptus grandis, Erythrina lithosperma and Cassia spectabilis did not inhibit spore germination and germtube elongation of E. vexans, in vitro when 5, 10 and 15% of their cell free extracts were used.

REFERENCES Andrews J.H. (1992). Biological control in the phyllosphere. Ann. Rev. Phytopathol. 30, 603-35. Arulpragasam P.V., Addaickan, S. and Kulathunga, S.M. (1986). Recent developments in the chemical control of blister blight leaf disease of tea. Effectiveness of EBI fungicides 1-12. Balasuriya, A. and Kalaichelvan, J. (2000). Is there potential in natural tea phylloplane microorganisms in the control of blister blight leaf disease of tea (Camellia sinensis)?. The planter 76(892): 409-17. Beattie, G.A. and Lindow, S.E. (1995). The secret life of foliar bacterial pathogens on leaves. Ann. Rev. Phytopathol. 33:145-72. Cappuccino, J.G. and Sherman, N. (1983). Microbiology: A laboratory manual, pp 465. Reading: Addison-Wesley publishing company. Fokkema, N.J. (1981). Fungal leaf saprophytes, beneficial or detrimental?. In: Microbial ecology of the phylloplane: 433-54 Blakeman JP, ed. London: Academic Press.

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Fravel, D.R., Connick, Jr. W.J. and Lewis, J.A. (1998). Formulation of microorganisms to control plant diseases. In: Formulation of microbial biopesticides, pp 187-202 Burges HD, eds. Dordrecht: Kluwer Academic Publishers. Gunasekara, T.S. (1994). Isolation and enumeration of microbial inhabitants of tea phylloplane. Sri Lanka J. Tea Sci. 63(1):19-27. Korsten, L., De Jager, E.S., De Villiers, E.E., Lourens, A., Kotze, J.M. and Wehner, F.C. (1995). Evaluation of bacterial epiphytes isolated from avocado leaf and fruit surfaces for biocontrol of avocado postharvest diseases. Plant Dis. 79(11), 1149-56. Lindow, S.E. and Mercier, J. (2000). Role of leaf surface sugars in colonization of plants by bacterial epiphytes. App. Env. Microbiol. 66, 369-74. Merrall, G.T. (1981). Physical factors that influence the behaviour of chemicals on leaf surfaces, In: Microbial ecology of the phylloplane, pp 265-81 Blakeman JP, ed. London: Academic Press. Ordish, G. (1952). Untaken harvest, pp171. London: Constable & Co. Ltd. Swinburne, T.R. (1981). Iron and iron chelating agents as factors in germination, infection and aggression of fungal pathogens. In: Microbial ecology of the phylloplane, pp 227-43 Blakeman JP, ed. London: Academic Press. TRI Advisory Circular, (2002). Serial No. , Tea Research Institute of Sri Lanka, Talawakelle, Sri Lanka.

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