(Conn and Franco 2004 ; Franco et al. 2007 ; Taechowisan et al. 2003 ). Previous
studies have demonstrated that endophytic actinobacteria were capable of
improving grain yields in the presence of common fungal root pathogens in a range
between 5 and 60% comparing with untreated controls (Franco et al. 2007 ).
Moreover, several actinobacterial species have been shown to be effective against
various soil-borne plant pathogens such as, Fusariumspp. (Cao et al. 2005 ;
Gopalakrishnan et al. 2011 ; Taechowisan et al. 2003 ),Pythiumspp. (Hamdali et al.
2008 ; Verma et al. 2009 ), Alternaria spp. (Chattopadhyay and Nandi 1982 ;
Vernekar et al. 1999 ), andRhizoctoniaspp. (Sadeghi et al. 2006 ; Sharma 2014 ),
being capable of protecting different important crops. Comparing with plant growth
promoting rhizobacteria, the use of endophytic microorganisms as microbial
inoculants for biocontrol strategies offers considerable advantages, since competi-
tion effects are greatly reduced in the colonization of the internal tissues of the
plant, thus increasing the chances of survival, growth, and effectiveness of the
endophytic inoculants (Coombs et al. 2004 ; Rosenblueth and Martinez-Romero
2006 ).
8.3 Isolation of Actinobacterial Endophytes
Endophytic actinobacteria may be isolated from a wide diversity of plants. Isolation
of these microorganisms is dependent on several factors, such as host plant species
and age, sampling mode, sampling season, cultivation conditions, surface steril-
ization strategy, and selective media used (Gaiero et al. 2013 ; Kaewkla and Franco
2013 ; Zhang et al. 2006 ). The selected isolation procedure will determine the
spectrum of endophytes recovered and should be able to yield the largest possible
number of endophytes, while at the same time eliminating epiphytic microorgan-
isms from the surface of plant tissues (Hallmann et al. 2006 ; Le et al. 2015 ; Li et al.
2012 ). Collected plants, or plant parts, should be processed as soon as possible
within a period of 24 h. Samples should be stored at 4 °C between sampling and
processing.
The critical step in the isolation of endophytic actinobacteria lies in the surface
sterilization of plant tissues. This may be achieved through the use of surface
sterilizing agents, with the most common ones being sodium hypochlorite (3–10%),
ethanol (70–95%) and hydrogen peroxide. Other less conventional sterilizing
agents, such as sodium chlorate (5%), sodium thiosulfate (2.5%), and sodium
bicarbonate (10%) have also been employed for the inhibition of growth of
endophytic fungi (Dochhil et al. 2013 ; Qin et al. 2008 ). The concentration of the
sterilizing agents will depend on the permeability of the plant tissues. In some
cases, sterilization efficiency is improved through the additional use of surfactants,
such as Tween 20, Tween 80, or Triton X-100, which reduce surface tension and
enable a better action of the sterilizing agent (Hallmann et al. 2006 ). Sterilization
protocols typically include a tissue washing step, to remove soil particles and
loosely adhered epiphytic microorganisms, followed by disinfection (which may or
166 M.F. Carvalho et al.