not be preceded by a pre-treatment step with a surfactant),final rinse with sterile
water, and sterility control to evaluate the sterilization efficiency. Apart from this
standard protocol, some additional strategies are available to increase the isolation
efficiency of endophytic actinobacteria. For instance, Qin et al. ( 2009 ) suggest the
use of a thiosulfate solution after the disinfection step with sodium hypochlorite to
minimize loss of endophytes caused by the presence of traces of disinfectant in
treated plants tissues. Nimnoi et al. ( 2010 ) suggested soaking treated plant samples
in a 10% NaHCO 3 solution in order to inhibit growth of endophytic fungi. Control
tests of sterilization efficiency often consist in plating a sample of water derived
from the last washing step or directly platting a surface-sterilized plant tissue.
Microorganisms can only be assumed to be endophytes if sterility control tests are
completely negative.
Ideally, the sterilization protocol should be adapted according to the plant spe-
cies, age and type of plant tissue. After the surface sterilization procedure, sterilized
plant tissues are inoculated in appropriate growth media, using one of two common
strategies: (i) tissues are aseptically cut into small fragments (Coombs and Franco
2003 ; de Oliveira et al. 2010 ; Sardi et al. 1992 ) or (ii) tissues are macerated with a
mortar and pestle (El-Tarabily et al. 2009 ; Garbeva et al. 2001 ; Hallmann et al.
2006 ; Kaur et al. 2015 ). In the latter case, in order to prevent inhibition of growth of
endophytic actinobacteria caused by plant enzymes or toxins released during the
maceration process, macerated samples may be diluted or buffered with appropriate
compounds such as phosphate buffer, polyvinylpyrrolidone or EDTA (Golinska
et al. 2015 ; Hallmann et al. 2006 ). More recent methods combining enzymatic
hydrolysis and differential centrifugation have been alternatively used and shown
very efficient in the isolation of endophytic microorganisms, especially rare
endophytic actinobacteria (Jiao et al. 2006 ; Qin et al. 2009 ).
The selection of growth medium is a very important step in the isolation of
actinobacterial endophytes. Nutrient poor media, such as tap water–yeast extract
agar (TWYE), humic acid–vitamin B agar (HV), and yeast extract–casein hydro-
lysate agar (YECD), have been reported to be very effective in the isolation of these
microorganisms (Coombs and Franco 2003 ; Qin et al. 2009 ). The formulation of
growth media with nutrients identical to those found in plants has also been shown
to be an effective strategy for the isolation of endophytic actinobacteria. Qin et al
( 2009 ) isolated rare endophytic actinobacteria comprising several genera using
growth media supplemented with the aminoacids L-asparagine, proline or arginine
as nitrogen sources and carbon substrates commonly found in plants, such as cel-
lulose, fucose, or xylan. Addition of plant extracts to the growth medium is another
effective strategy (Qin et al. 2011 ). Growth media should be supplemented with
antibiotics such as nystatin, nalidixic acid, or cycloheximide (50 or 100μg/ml) to
inhibit growth of fungi and Gram-negative bacteria (Golinska et al. 2015 ; Lee et al.
2008 ; Qin et al. 2011 ). Examples of isolation strategies used for the recovery of
endophytic actinobacteria from various plants with agricultural relevance are pre-
sented in Table8.1.
8 Endophytic Actinobacteria for Sustainable Agricultural Applications 167