Agriculture Reference
In-Depth Information
Surface Disinfection and Maceration
As reviewed by Hallmann and others (1997b), the fi rst methods to identify endophytes
in plants were based on various surface decontamination strategies. Some commonly
used chemical sterilants include sodium hypochlorite (NaOCl, 2-10%), ethanol (70-
90%), hydrogen peroxide (H
2
O
2
, 3%), peracetic acid (0.03%), and mercuric chloride
(HgCl
2
, 0.1%), with two or more of these sterilants sometimes used in combination.
In order to increase disinfection effi cacy, these aqueous solutions can be supplemented
with detergents such as Tween 20 or 80 to reduce the surface tension on plant surfaces.
After a treatment period of 10 sec to 10 min—depending on the chemical disinfectant
used—the plant tissue is repeatedly rinsed in sterile water or buffer to remove any
residual disinfectant and then dried with a sterile tissue or in air under ambient condi-
tions. Alternatively, plants with tap roots and stems can be dipped in ethanol and
fl amed for surface disinfection (Yan and others 2003). Selection of disinfectants and
treatment times depends on the plant species (e.g., woody, root crop, leafy green, fruit,
berry), maturity (e.g., seedlings, young or old plants), portion of the plant (e.g., roots,
stems, leaves, seeds) and surface topography (e.g., waxy, smooth, wrinkled). Therefore,
surface disinfection needs to be optimized for each specifi c situation. Ideally, surface
disinfection will eliminate all epiphytic bacteria as confi rmed by sterility testing
(Hallmann and others 1997b), with the entire endophytic population remaining both
viable and recoverable after treatment. However, this end result is seldom, if ever,
achieved, with all of these treatments yielding varying percentages of surviving epi-
phytes and inactivated endophytes. Two reviews (Lodewyckx and others 2002; Stone
and others 2000) highlighted the methods used to isolate and characterize endophytic
bacteria from different plant species. With respect to foodborne pathogens, internaliza-
tion of
Salmonella
and
E. coli
O157:H7 in fresh produce has been demonstrated using
various surface sterilization methods including sodium hypochlorite for apples
(Buchanan and others 1999), ethanol fl aming for tomatoes (Ibarra-Sánchez and others
2004 ), HgCl
2
and ethanol for sprouts (Dong and others 2003; Itoh and others 1998),
and sodium hypochlorite for lettuce and spinach (Jablasone and others 2005;
Johannessen and others 2005; Solomon and others 2002). To quantify endophytic
bacteria, surface-sterilized plant tissue is macerated either manually (e.g., mortar
and pestle) or mechanically (e.g., blender, stomacher, tissue pulverizer) in a buffer
solution, with serial dilutions and then plated to appropriate media.
To avoid the disadvantage from strong surface disinfectants that may penetrate and
kill endophytes, additional alternatives are needed. Interestingly, some potentially
useful methods routinely used to recover bacteria from food contact surfaces including
agitation with or without glass beads, votexing, sonication, and pulsifi cation have not
been widely examined and warrant further investigation.
Extraction of Endophytes
Alternatives to surface disinfection include vacuum and pressure extraction. The
vacuum technique used in plant pathology to extract fastidious bacteria from xylem
is also useful for recovering endophytes from grapevines and citrus fruits. The
Scholander pressure bomb method (used to measure plant respiration in aliquots
forced out from plant tissue under high pressure) was used to study endophytes
in cotton, soybeans, and beans (Hallmann and others 1997a). Compared to other
