STEM COLONIZATION OF PEA CULTIVARS BY INDIGENOUS ENDOPHYTIC BACTERIA M ELVIRA-RECUENCO and JWL VAN VUURDE DLO Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands Background and objectives Bacterial endophytes have been defined as bacteria which can be isolated from surface disinfested plant tissue or extracted from inside the plant and which do not visibly harm the plant[1]. The presence of endophytic bacteria in healthy plant tissue has been reported for many plant species and plant parts at various stages of growth, including seeds and pods of peas[2]. Due to the beneficial effects of endophytic bacteria on plant growth promotion and reduction of disease symptoms shown in recent studies[l], research on these bacteria has become increasingly more important. The purpose of this paper was to study the presence of endophytic bacteria in the stem of various pea cultivars when grown under field conditions, analysing bacterial types and populations. Materials and methods Collection of symptom-free pea plants from field trials was made in collaboration with Nunhems Zaden B.V., Haelen, The Netherlands. Plants were cut above ground level (second node). 1. Screening of cultivars Eleven cultivars, ten plants per cultivar from five field repetitions, were sampled at the flowering stage. Stems were surface disinfested with 1% available chlorine plus 0.1% Tween 80 during 5 minutes and rinsed 3 times in sterile distilled water. Transections along the stem were cut off with an ethanol flamed scalpel and four prints per transaction were made on 5% TSBA plates. 2. Endophytic colonization of the cultivar Twiggy Twenty plants of the cultivar Twiggy from four field repetitions were sampled at pod stage. Transections along the stem were made as described above. Stem parts (the third and the fourth internode) were homogenized and the extract was plated on 5% TSBA, R2A and SC media by spiral plating. Results and conclusions Cultivar differences in bacterial colonization were observed, being Twiggy the cultivar which showed the highest colonization. Populations varied from 103 to 107 CFU/g fresh stem tissue which is in agreement with the range described for other crops [3]. Populations in one of the field repetitions of the cv. Twiggy were 102-103 higher than the rest. Bacterial identification was made by fatty acid analysis (Plant Protection Service, Wageningen, The Netherlands). Two predominant bacterial types were found, Pseudomonas fluorescensand Erwinia herbicola. Other types were Pseudomonas viridiflava and Bacillus megaterium. The last one was only isolated from the field repetition where total bacterial populations were higher. Variation between media was very low, giving similar counts and types for the three media tested. Populations decreased acropetally along the stem as for sweet corn and cotton[3]. Prints of transactions from surface disinfested stem parts proved to be an efficient tool for the presence and semi-quantitative estimation of endophytic bacteria The results show that the stem of the pea plant can be colonized by indigenous endophytic bacteria which may be affected by cultivar properties and local conditions. References 1. Hallmann J, Quadt-Hallmann A, Mahaffee, WF, Kloepper, JW, 1997. Canadian Journal of Microbiology 43(10), 895-914 2. Samish Z, Etinger-Tulczynska R, Bick M, 1963. Journal of Food Science 28, 259-266 3. McInroy JA, Kloepper JW, 1995. Canadian Journal of Microbiology 41, 895-901