Microbial Diversity of Popular Arabica and Robusta Selections

K. Velmourougane, D. Praffulla Kumari, C.B. Prakasan and Jayarama

Introduction

As coffee is a perennial crop, it is vital to understand the biological factors influencing the growth, development and quality. One of most important factor which directly influences the soil fertility, plant development and coffee quality is microbial activity, microbial association and micro bial interactions with coffee ecosystem. Since microbes are involved in soil formation, nutrient mobilization and uptake, disease resistant and quality attributes in any plant system, it is essential to understand their association and distribution in soil ecosystem.

In India, there are different selection~ developed in arabica and robusta coffee for their yield characters, pest and disease resistant and quality attributes. There are several reports on performance of different selections of arabica and robusta in different coffee growing regions on yield, resistance to pest and diseases and quality. Though soil type, elevation, rainfall, fertilization, cultivation practices etc. influence the plant development and quality, there are wide variations among the different selections of coffee to the above mentioned factors. The variation could be mainly due to the soil biological property i.e. microbial activity and their interactions with the host plant in addition to physical and chemical properties.

Hence better understanding of the soil microbiological properties of coffee ecosystem could useful in development of better integrated nutrient, pest and disease management strategies for coffee in future. With the above objective, studies have been undertaken at Coffee Research Sub Station, CRSS, Chettalli to study the microbial distribution and association with prominent selections of arabica and robusta coffee from soil to beans. The salient findings of the study are presented in this paper.

Materials and methods

Samples of bulk soil (0-9"), rhizosphere soil (0-9") phyllosphere (Leaf surface), caulosphere (Bark), green developing berries and ripe coffee fruits of prominent selections of arabica (Sln-795, Sln-7.3, Sln-9, Sln-5B, Sln-ll and Sln-12) and robusta coffee (Old robusta, Sln274 and CXR) were collected from Coffee Research Sub Station Farm, Chettalli, Kodagu, Karnataka. Microbial population of bulk soil, rhizosphere soil and caulosphere were enumerated using standard dilution plate count method, while microbes associated with phyllosphere were enumerated adopting leaf washing technique. External microflora of developing berries and ripe fruits were assessed by standard serial dilution technique.

Microbial counts were made on selective media (Nutrient agar for bacteria, Czapeks agar for fungi, Ken knight agar for actinomycetes and buffered yeast agar for yeast) after decimal dilution of soil samples (104) using sterile 0.1 % peptone water by spread plate method and the plates were incubated at optimum temperature in triplicates. The functional/ physiological groups of microbes (pseudomonas sp, Phosphobacteria, Pectionolytic microbes, Cellulolytic microbes, Chitinolytic microbes, proteolytic microbes, starch hydrolytic and Azotobacter sp) were enumerated by following standard microbiological methods mentioned in "Methods of soil analysis-Part2 Chemical and microbiological properties (1982)". The colonies that appeared after incubation period were counted as Colony Forming Units (Cfu)/g of sample. The colony characteristics were observed and representative single colonies were isolated and sub cultured on respective media. Cell morphology was observed microscopically after staining. All the bacterial cultures were identified according to Bergey's Manual of Determinative Bacteriology (Holt et aI, 1994). Identification of yeast and fungi were done as per the Manual "Illustrated genera of imperfect fungi" (Barnett, 1960).

Bean mycological Analysis: The coffee samples from different selections were surface sterilized with 1 % sodium hypo chloride for about 10 minutes. The sodium hypochlorite treated clean coffee samples were washed thrice with sterile distilled water, wiped with sterile filter paper and then aseptically plated on the sterilized DG-18 medium (Hocking and Pitt, 1980) and Czapeks agar. For each sample, a total of 70 beans (7 beans/plate) were plated to study the extent of mould infection. The inoculated plates were incubated at 25°C and observations were made after 5-7 days of incubation. The mould infection was expressed as bean infection rate (%) (All the . values represent frequency, not growth. Infections are considered independently so the sum of infections, taxon-by-taxon, can exceed 100% overall bean) and representative colonies of mould species presumptively identified according to the manual of Klich and Pitt (1988) and "Illustrated genera of imperfect fungi" (Barnett, 1960). Results and discussion

The total microbial population of different arabica selections is presented in Table-t. In bulk soil, the higher microbial population was recorded in Sln-12 (156 cfu x 104) follwed by Sln-9 (78 cfu x 104) andSln-11 (64 cfu x 104). The lowest microbial population of 40 cfu x 104 was recorded in Sln-795. In rhizosphere, higher microbial count was recorded in Sln-12 (176 cfu x 104) followed by Sln-795 (132 cfu x 104) and the lowest count was observed in Sln-7.3 (64 cfu x 104). Sln-795 recorded higher fungal (7 cfu x 104) and actionomycetes count (32 cfu x 104) in rhizosphere. In phyllosphere (Leaf surface), Sln-9 recorded higher population (90 cfu x 104) followed by Sln-5B (69 cfu x 104). Higher fungal count of 16 cfu x 104 and 14 cfu x 104 was recorded in Sln-5B and Sln-9 respectively in phyllosphere. In caulosphere (Stem surface), very high population of 163 cfu x 104 was recorded in Sln-5B followed by Sln-12(97 cfu x 104). In developing green berries, higher microbial count of 121 cfu x 104 was recorded in Sln-5B followed by Sln-7.3 followed by Sln-5B Sln.73 (65 cfu x 104). In ripe fruits, higher microbial load of 119 cfu x 104 was recorded in SIn. 7.3 followed by by Sln.513 (78 cfu x 104). From the samples analysed, it is found that higher microbial population was recorded in Sln-12 followed by Sln-5B and Sln-9 in arabica selections .

In robusta, three selections were studied for the microbial association and were presented The higher microbial population of 75 cfu x 104 was recorded in SIn CXR followed by 54 cfu x 104 in Sln-274 in bulk soil. In rhizosphere, higher microbial count was recorded in Sln CXR (117 cfu x 104) followed by Sln-274 (Fig-2). Interestingly higher fungal counts were recorded in robusta rhizosphere compared to arabica rhizosphere. Old robusta recorded higher phyllosphere and caulosphere count (52 & 68 cfu x 104) followed by Sln-274 (37 & 44 cfu x 104). In developing berries and ripe fruits, higher association was observed in SIn CXR (51 & 83 cfu x 104) followed by Sln-274 (41 & 65 . cfu x 104). In robusta, in general, higher microbial association wasFor better understanding the microbial activity, association and interactions in coffee soil, R:S ratio has been worked out and the data are presented in Table-2. In arabica selections, higher R:S ratio was observed in Sln-795 (3.3:1) followed by Sln-9 (1.62:1). The lowest R:S ratio was observed in Sln-12 (1.13:1). In robusta, higher R:S ratio was recorded in Sln-274 (1.74:1) followed by old robusta (1.65:1).

The data on functional groups of microbes associated with different arabica and robusta selections were presented in Table-3, Fig 1 & 2. In general, higher functional groups were recorded on Sln-5B (79 cfu x 104) and SIn CXR (56 cfu x 104) in arabica and robusta respectively. In arabica higher Pseudomonad's was recorded in Sln.5B (56 cfu x 104) followed by Sln.9 (43 cfu x 104). In robusta higher Preudomonad's was recorded in SIn. CXR (32 cfu x 104) followed by old robusta (28 cfu x 104). Higher Phosphobacterial count of 4 cfu x 104 was recorded in Sln-7.3 of arabica and 2 cfu x 104 in old robusta. There is no much variation in populations of Azotobacter, Pectinolytic, cellulolytic and chitinolytic microbes among different selections of arabica and robusta. Interestingly, higher proteolytic microbes were recorded in arabica selections, while robusta selections recorded higher starch hydrolytic microbes. The data on qualitative distribution of microbes among different arabica and robusta selections is presented in Table-4. In general, Aspergillus spp. was found to be the dominant soil fungi in all the arabica selections studied. While robusta, Fusarium spp. found to be the dominant soil fungi followed by Aspergillus spp. In phyllosphere and caulosphere, Cladosporium spp. was found to be the dominant mycoflora in both arabica and robusta followed by Fusarium spp. in arabic a selections and Aspergillus spp. in robusta selections. In developing berries and ripe fruits, Cladosporium spp. found to be the dominant followed by Fusarium spp. in both arabica and robusta selections, while higher yeast counts were recorded in ripe fruits compared to green berries.

The data on mycology (fungal association) of coffee beans from different arabica and robusta selections is presented in Table-5. In general higher total mould infection in dried coffee beans was recorded in SIn -7.3 (79%) followed by Sln-11 (73%) and the lowest fungal infection of 39% was recorded in SIn - 5B in arabica. In robusta, higher mould infection was recorded in old robusta (83%), while SIn CXR recorded lower fungal infection (59%). In arabica, Aspergillus spp. was found to be the dominant seed borne fungi bollowed by yeast and Fusarium spp. while Aspergillus Nigar was found to be the dominant fungi in robusta selections followed by Aspergillus spp. and Fusarium spp. Higher yeast infection of 33% and 37% was recorded in Sln-5B in arabica and SIn CXR in robusta respectively. The findings of the present study are in accordance with similar observations made by Velmourougane et al (2000a & 2000b) in arabica coffee in Chikmagalur region, Karnataka.

References:

Barnett, H.L. 1960. Illustrated genera of imperfect fungi. 2nd Ed. Minneapolis. Burges. 225p.

Hocking.A.D. and PitU.I.1980. Dichloran-glycerol medium for ~numeration of xerophilic fungi from low moisture foods. Appl. Environ. Microbial. 39, 488-492.

Holt. J.G., Krieg. N.R, Sneath. P.H.A., Staley. J.T. and Williams. S.T. 1994. Bergey's Mannual of Determinative Bacteriology. 9th edn.pp.529-566. Baltimore Williams & Wilkins.

Klich. M.A. and Pitt. J.I. 1988. A laboratory guide to common Aspergillus sp and their teleomorphs. CSIRO Division of Food processing, Sydney. 166pp.

Methods of soil analysis-Part-2Chemical and Microbiological properties, 2nd Ed, Ed by : A.L. Page., RH. Miller and D.R Keeney, 1982.

Velmourougane. K., Panneerselvam. P and R.P.A. Alwar. 2000ao Qualitative and quantitative distribution of microflora associated with coffee plants and berries. In: Recent Advances in Plantation Crops Research. pp 396399.

Velmourougane. K, Panneerselvam. P, Shanmukhappa. D.R., Gopinandhan. T.N., Srinivasan. C.S. and R. Naidu. Study on micro flora associated with high and low grown coffee of arbica and robusta. J. Coffee Res. 28 (1&2) 9-19, 2000bo