CHARACTERIZATION OF ENDOPHYTIC BACTERIA ISOLATED FROM HEALTHY AND DISEASED COFFEE TREES SHOWING WITCHES BROOM SYMPTOMS UNDER FIELD CONDITIONS IN PUERTO RICO 1 2 Carlos Bolaños C. and Mildred Zapata 1
Graduate Student, 2 Professor
Crops and Agro-Environmental Sciences Department, University of Puerto Rico – Mayagüez Campus, PR.
[email protected]
SUMMARY Coffee (Coffea arabica L.) is an economically and socially important crop, in 2009 this industry generated revenues of $ 35 millions in Puerto Rico. Main production problems are of biotic origin. Among bacterial diseases affecting this crop, coffee leaf scorch (CLS), caused by the bacterium Xylella fastidiosa Wells et al., is significantly important in many countries due to economic losses. In February, August, November and December of 2010, samples were collected from coffee trees showing marginal and apical leaf scorch, yellowing of new leaves, reduction of internode lenght, abnormal production of new flushes (witches broom). Isolations on PW medium were made from branches and leaf veins, to identify endophytic bacteria associated with the symptom expression. Strains were purified and tested for reaction to catalase, oxidase, gram stain, and endospore formation, and growth on TSA medium. DAS ELISA for Xylella fastidiosa was performed for all gram negative bacteria. Cluster analysis indicated that twice as many Gram negative bacteria of fastidious growth (PW +, TSA –) were found in diseased versus healthy trees.
INTRODUCTION
METHODOLOGY
Endophytic bacteria reside within plant tissues, three of them (Xylella fastidiosa, Clavibacter xyli and Pseudomonas syzygii) have been characterized like fastidious xylem limited bacterial plant pathogens. In Puerto Rico, several coffee plants were observed with symptoms like marginal and apical leaf scorch, yellowing of new leaves, reduction of internode lenght, abnormal production of new flushes (witches broom). In this study, we tried to identify and characterize the bacterial species related to the vascular tissues of diseased coffee plants in Puerto Rico.
Field collections of symptomless and symptomatic plant material were made in 2010 in coffee farms of the municipalities: Las Marías (18°13’14’’; W66°01’38’’), Adjuntas (18°09'30'',W66°45'27''), Yauco (N18°09’57’’; W66°49’36’’) and Jayuya (N18°09’35’’; W66°38’45’’). Samples were collected during the rainy (August and November) and dry (December and February) seasons. To isolate the endophytic bacteria, few branches and leaf veins were soaked in sodium hipoclorite (0,25%) and ethanol (70%), rinsed with destilled water and placed in vials with periwinkle (PW) broth medium. The vials were placed in the refrigerator overnight at 4°C, to increase the number of endophytic bacteria released into the broth. A sample of 100ul of the liquid broth were dispensed on PW solid medium ( Soytone 4g/l, Triptone 0,5g/l, K2HPO4 0,6g/l, KH2PO4 0,5g/l, MgSO4.7H2O 0,4g/l, (NH4)2HPO4 0,8g/l, Potato starch 2g/l, L-Histidine HCl 1g/l, Agar 12g/l, L-glutamina 4g/l, Bovine serum albuminum 3g/l, Hemin chloride 10mg/l, Phenol 20mg/l) amended with Cycloheximide 50mg/l, to avoid fungal growth. Plates were incubated at 28°C up to one month. Colonies were characterized for the following traits: size, form, margin, elevation, color, ability to grow on tryptic soy agar (TSA), gram reaction, ability to form endospores, oxidase and catalase reaction. DAS ELISA was carried out using Xf pathoscreen kit (AGDIA, Elkhart, IN), for all gram negative bacteria. Figure 3. Cluster analysis using single linkage for: Gram- TSA- (left above), Gram – TSA+ (Right above), Gram + TSA+ (Left below) and Gram + TSA – (Right below)
b
c
a
Figure 1. Field Colletions: (a )Yellowing of young leaves on coffee tree (b) Abnormal production of new flushes forming a “witch’s broom” (c) Geo-reference of samples using ESRI-Explorer ARC GIS.
a
b
RESULTS Cluster analysis (Figure 3) was carried out using nearest neighbor method, and the calculated distance was the Euclidean with normalized data (Infostat, 2006). All clusters showed cophenetic correlation higher than 80%. Seven strains belong to the group of fastidious growth with gram negative reaction (4853, 5011, 4986, 5221, 5151, 5040 and 4986). Only two of these strains (5011 and 5151) were obtained from symptomless tissues, the other strains were obtained from symptomatic plants, it represents approximately two times higher. The most common configuration obtained were catalase positive, oxidase negative, and able to growth on TSA media. The averages DAS ELISA readings for vein extract of the plants was 0.49, and for all gram negative strains tested was 0.06, compared with 0.03 for the blank (only general extraction buffer GEB).
d c
Figure 4. Strain morphology (a 5221, b 5040, c 5151, d 5029) on PW medium.
DISCUSSION Plants normally, are associated with a broad and diverse population of microorganisms. Their interactions could be beneficial, neutral or harmful. In this study, 32 strains were isolated from coffee leaf veins and branches, with symptoms like: witches broom, leaf scorch, yellowing, and 28 bacterial strains from symptomless plants. Eighty percent of all isolates grew in the first 1 to 5 days post inoculation. We observed that gram negative bacteria of fastidious growth were two times higher in symptomatic plants versus symptomless plants. There are a lot of strains isolated from coffee veins and branches; the use of harmless, naturally occurring endophytic bacteria to compete with Xylella could be a measure of biological control. Therefore, these strains could be potential antagonistic agents against Xyllela fastidiosa. REFERENCES: Infostat. 2006. Infostat, versión 2.0. Grupo Infostat, FCA, Universidad Nacional de Córdoba. Córdoba, Argentina. Lima, J., V. Miranda, J. Hartung, R. Brlansky, y E. Carlos, 1998. Coffee Leaf Scorch Bacterium: Axenic Culture, Pathogenicity, and Comparison with Xylella fastidiosa of Citrus. PlantDis82:94-97.
Figure 2. ELISA test for Xylella fastidosa, using coffee leaf veins and branches: Rows from left to right are presented with readings at 650nm aborbance (in parentheses) Rows: 1E(0,411); 1H Negative control (0,056); 2H Positive control (2,371); 3A (0,306); 11C (0,905); 11D (0,494); 12D (0,466); 12G (0,423); 12H (0,399).
Marino-Cardenas, J., 2008. Identificación de bacterias en cicadélidos vectores potenciales de Xylella fastidiosa Wells et al., que se alimentan del tejido vascular de plantas de café Coffea arabica L. en tres localidades de Puerto Rico. Tesis M.S. Universidad de Puerto Rico. Recinto Universitario de Mayagüez. 160pp Marino- Cardenas J., y Zapata, M., 2009. Diversidad de bacterias en cicadélidos vectores potenciales de Xylella fastidiosa Wells et al. en cafetales en Puerto Rico. J. Agric. Univ. P.R. 93 (1-2). 101-121p.
ACKNOWNLEDGEMENTS: USDA Proyect ZTS 51 Potential effects of Xylella fastidiosa Wells et al., on shade coffee establishment in Puerto Rico.
