The coffee plantation is mainly observed in the Nilgiris, but it also grows in the Eastern Ghats in Andhra Pradesh. This area has high humidity and temperature and as a result of which there is high incidence of rust disease. But when the leaf fragment was aseptically transferred to nutrient agar medium, the bacteria grew and the fungi did not develop because of its obligatory parasitic nature. Opaque, ash-white, mucoid and rhizoidal colonies with or without yellow fringe were obtained indicating a possible bacterial infection. The bacteria was isolated and sub-cultured for identification and characterisation purposes. It was identified as a rod-shaped, gram negative, aerobic bacterium giving a positive catalase test and negative for oxidase, indole production, methyl red, voges proskauer, citrate utilisation and nitrate reduction tests. It also showed no gas production from lactose. Hence, the organism was identified to be Pseudomonas syringae of Pseudomonadaceae family which is the causal organism of the leaf blight disease in plants. However, the symptoms of Leaf Blight are not observed in the coffee leaves indicating that the pathogen can multiply on the surface of the healthy plant and provide inoculum even in the absence of the disease. This is a classic example of lack of quantitative relationship between pathogenic populations and disease incidence.
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Coffee is one of the most important plantation crop grown mostly in South Indian states at a height between 750-2000 meters above MSL. The coffee plantation suffers from a number of diseases of which coffee rust is one. This disease was first reported from Sri Lanka in 1868 and the following year from the state of Karnataka. The rust organism-Hemileia vastatrix, a fungus of the order Uredinales mainly attacks the leaves and is rarely found on young stems and fruit. Typically, the disease is recognized by the yellow-orange powdery lesions or spots on the underside of leaves. Initially, very young lesions appear as chlorotic or pale yellow spots before sporulation is evident. These spots vary in size and can coalesce during their development. Since sporulation of this pathogen occurs through the stomata, lesions characterized by the ruptured epidermis of most rusts do not occur, and the lesions are not referred to as pustules. Coffee serves as the obligate host of coffee rust, that is, the rust must have access to and come into physical contact with coffee (Coffea arabica) in order to survive. Coffee leaf rust is spread by wind and rain from spores present in the lesions on the underside of the plant.(Mitchell 1984)
Generally we do see some organisms growing singly or in combinations and very often they exhibit interaction or hyperparasitism, resulting in the modification of the disease cycle. While we were trying to isolate and study the rust fungi from the coffee leaves we collected from the coffee plantations in the Eastern Ghats in Andhra Pradesh we obtained a bacterial culture instead of the fungi. The fungus does not grow in artificial media as it is an obligate parasite. Hence we tried to characterise and study the bacteria obtained from the rust infected coffee leaves.
Materials and Methods
The bacterial culture obtained from the Potato Dextrose Agar slants inoculated with the diseased leaf portions was sub cultured in 5 Nutrient Agar slants along with inoculation of a fresh batch of Nutrient Agar slants with the infected leaf after surface sterilization with 1% HgCl2.
Gram characterization of the bacteria was done. Also the size was measured. The antibiotic sensitivity against Streptomycin, Tetracycline, Ampicillin and Linezolid was tested using the disc diffusion method (Kirby-Bauer, 1960) for both fresh culture and subculture of the bacteria.
For identification and characterization of the bacteria the standard biochemical tests were performed namely Oxidase test(W.L. Gaby, C. Hadley, J. Bact., 74, 365 (1957)), catalase test, indole production test, Methyl Red test(Michaelis and Marcora, 1912), Voges Proskauer Test(O. Voges, B. Proskauer, (1898)), Citrate utilization test(Koser, 1923-24), Nitrate Reduction Test and lactose fermentation test. The results were tabulated and organism was identified.
Results and Discussion
The organism was found to be a rod shaped Gram-negative bacterium. Its average length is about 3 micron and breadth is around 0.5-1 micron. It was seen from the antibiotic sensitivity test performed with this pathogen that Streptomycin was most effective against it followed by Tetracycline which showed a smaller zone of inhibition (Refer Table 1). The organism gave a positive Catalase test and tested negative for Oxidase, Indole production, Methyl Red, Voges Proskauer, Citrate utilization and Nitrate reduction tests. It showed no gas production from Lactose in the Lactose fermentation test (Refer Table 2).
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On the basis of all these biochemical tests the organism was identified to be Pseudomonas syringae belonging to the genus Pseudomonas. Based on 16S rRNA analysis, it has been placed in the P. syringae group. It is a prolific plant pathogen which can infect a wide range of plant species, and exists as over 50 different pathovars. It is named after the lilac tree (Syringa vulgaris), from which it was first isolated. Unlike the other species of pseudomonas it gives a negative oxidase test.
A particular strain of Pseudomonas syringae which normally causes bacterial blight in a wide range of plant species also infects coffee plants though not always resulting in the disease incidence as seen in this case. P. syringae is a nutritionally versatile organism. Although it is a plant pathogen, it can also live as a saprotroph in the phyllosphere when conditions are not favourable for disease. This pathogen can multiply on the surface of the healthy plant and provide inoculum even in the absence of the disease. In the coffee leaves we got from the Eastern Ghats near Vishakhapatnam symptoms of leaf blight were not seen though the organism was found to be residing in the plant thus showing a lack of quantitative relationship between pathogenic populations and disease incidence. Some saprotrophic strains of P. syringae have been used as bio control agents against post-harvest rots.