# Endophytic Entomopathogenice Fungi: A Valuable Biological Control Tool against Plant Pests
## 3 Artificial Plant Inoculation with Endophytic EPF  
The artificial establishment of EPF in plants for endophytic colonization has also been examined for pest control [3; 68; 69]. More specifically, endophytic colonization can offer systemic plant protection against pests as the latter are negatively affected by the chemical changes triggered in the plant by the endophyte as well as by the secondary chemicals secreted by the fungus [56; 70; 71]. However, not all fungi are able to colonize plants--at least not for their whole life cycle--because they cannot adapt to the nutrient content inside the plant [72]. Nevertheless, a transient fungal/plant association can exist for a few days following a spray application because of the abundance of propagule on the leaves [44; 73].  
Endophytic EPF colonization in plants is influenced not only by soil microorganisms but also by environmental factors such as temperature and relative humidity [74]. Other factors that have been proven to play a vital role in the inoculation of EPF into plant tissues are the age and species of the plant, the growth medium, the conidial density and species of the EPF and the applied inoculation method [74; 75; 76; 77; 78; 79].  
Various methods and experimental protocols have been used to artificially inoculate endophytic EPF into crop plants. These methods can be categorized as: (a) spraying leaves with conidial suspension, (b) soaking seeds in conidial suspension, (c) injecting fungi inoculum into stems, (d) dipping of seedling roots in conidial suspension, and (e) soil drenching with conidial suspension. Table 11 in Appendix A summarizes previous studies where EPF have been successfully inoculated into the plant tissues of major crop plants.  
The foliar application is the most common method, with many promising results (Table 11). However, certain drawbacks have been reported and must be taken into serious consideration. The foliar endophytes can reduce the insect population by producing alkaloids that are toxic [53; 54]. The major concern of this method is the extremely localized colonization that is often limited to the foliar parts of the plant, with EPF being absent from stems and roots [77; 80]. Apart from that, poor efficiency of hyphal penetration into leaf tissues has been reported [76; 79], possibly due to the low density of stomata (natural entries for fungal infection), leaf surface structure and specific cuticular components.  
Soaking seeds in conidial suspension before propagation is another inoculation method that has been successfully applied to many major crops (Table 11). However, there have been reports where inoculation through the seed resulted in some or no colonization of the stem or leaf [77; 81]. This was attributed to the negative effect of soil microorganisms that may act antagonistically toward the EPF.  
Stem injection has also been evaluated as an inoculation method of EPF on various plants [74; 76]. When _B. bassiana_ was inoculated with this method, the highest post-inoculation recovery was yielded in coffee seedlings, compared with foliar spraying or soil drenching [76], and efficient _H. armigena_ control was provided for tomato plants [81].  
Dipping roots in conidial suspension has proven an effective inoculation method, although results were often contradictory when compared with other methods such as foliar application or seed treatment [79; 82]. The success of this method has been reported to be greatly dependent on the plant species.  
The soil drenching technique includes the watering of seedlings with conidial suspension. Similar to root dipping, the low colonization rate that is often recorded with this method has been linked to the interaction between EPF and other competing soil microorganisms. The use of sterile growth media instead of non-sterile soil significantly enhanced the success of this method [77].