First Report of Bacterium and Yeasts Associated with Pineapple Fruit Collapse in Espírito Santo State, Brazil.

In rainy and warm periods of the year, after a dry, hot season (December/March), commercially grown pineapple (Ananas comosus var. comosus) in Espírito Santo State, Brazil has been affected by fruit collapse disease with significant commercial losses (15 to 20%) each year. Symptoms include intense flesh fermentation, spontaneous exudation of liquid and froth, and ripe inner fruit tissue deterioration on plants and postharvest. Isolation of microorganisms of diseased fruits on nutrient agar, potato dextrose agar, and yeast extract peptone dextrose media consistently resulted in the recovery of a bacterium and three different yeasts. Koch's postulates were fulfilled by syringe inoculation of different concentrations of each microorganism (2.0 × 10⁵ to 2.0 × 10⁹ CFU/ml) singly and the organisms in combinations (only bacterium, only the yeasts, one yeast and the bacterium, two yeasts and the bacterium, and three yeasts and the bacterium) into disinfected (0.5% NaOCl) postharvest ripe pineapple fruits (cv. Pérola). Inoculated fruits were kept at 40°C for 5 days. Six fruits were used for each treatment and the experiment was repeated three times. Characteristic symptoms only occurred when all three yeasts and the bacterium were inoculated in combination at all inoculum concentrations. Each microorganism alone could not produce symptoms. Control fruits, inoculated with sterile water, did not develop disease symptoms. Cultures of each isolate were obtained and identified by morphological, physiological, biochemical, and genetic analyses. Molecular characterization by ribosomal sequence analyses of bacterium (16S rDNA) and yeasts (D1/D2 region of 26S rDNA) used universal PCR primers for bacteria (F968 and R1401) and yeast NL1 (5' GCA TAT CAA TAA GCG GAG GAA AAG 3') and NL4 (5' GGT CCG TGT TTC AAG ACG G 3'). Sequences were compared with those in GenBank. On the basis of the results, the bacterium was identified as a Klebsiella sp. and the yeasts as a Candida sp., Saccharomyces sp., and a Kloeckera sp. The bacterium was negative for protease, cellulase, and pectinase activity in the qualitative tests. We concluded that the combination of a Klebsiella sp. with the three yeasts, Candida sp., Saccharomyces sp., and Kloeckera sp., was responsible for the symptoms of pineapple fruit collapse. Symptoms of pineapple fruit collapse are similar to yeasty fermentation reported previously (2,3). These reports did not consider the interaction of yeast and bacteria. No relationship between the pineapple cultivars and the pathogens from specific field sites was found. Disease outbreaks seem related to naturally occurring fruit translucency, a physiological disturbance correlated with calcium, potassium, and nitrogen balance (1,3), which increases fruit cell-wall hydrolases and membrane permeability. This condition releases nutrients from the fruit and favors microbial growth. To our knowledge, this is the first report of a combination of bacterium and yeasts associated with collapse disease of pineapple in Brazil. Information on the pathogens responsible for collapse disease epidemics in Espírito Santo fields will be useful in breeding and disease control strategies. References: (1) R. P. Haff et al. J. Food Process. Preserv. 30:527, 2006. (2) C. Py et al. The Pineapple: Cultivation and Uses. Larose, Paris, 1987. (3) K. G. Rohrbach and M. Johnson. The Pineapple: Botany, Production and Uses. D. P. Bartholomew et al., eds. CABI Publishing, Wallingford, UK, 2003.