Morris CE, Monier JM, Jacques MA
INRA, Station de Pathologie Vegetale, Domaine St. Maurice, 84143 Montfavet cedex, France.
Appl Environ Microbiol. 1998 Dec;64(12):4789-95. doi: 10.1128/AEM.64.12.4789-4795.1998.
The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.
最近已证实陆地植物叶际存在微生物生物膜，但用于研究与活植物组织相关生物膜的技术却很少。在此，我们报告一种技术，用于估计叶片上以生物膜形式聚集的细菌群体比例，并从叶际微生物群落的生物膜和非生物膜（单独存在）成分中定量分离细菌。该技术基于通过轻柔冲洗从叶片上去除细菌，通过过滤分离生物膜细菌和单独存在的细菌，以及通过超声处理使生物膜解体。对该技术所用的过滤器进行了评估，测定其对单独存在细菌的非特异性保留率，以及在存在生物膜和其他颗粒的情况下对不同浓度单独存在细菌的过滤效率。还评估了此处所用超声处理条件的致死率和解体效率。通过该技术分离和定量细菌是基于使用培养基。然而，寡核苷酸探针、血清或落射荧光染料也可用于直接鉴定通过该技术产生的悬浮液中的生物膜细菌和单独存在的细菌。叶际群落生物膜丰度估计的初步结果表明，在阔叶菊苣和欧芹叶片上，生物膜中的细菌占细菌总数的10%至40%左右。
