(Ezra et al. 2004 ; Hallmann et al. 2007 ; Rashid et al. 2012 ; Kusari et al.2009a,
2013 ,2014a) followed by counter-check methods ensure proper sterility (Schulz
et al. 1998 ;Sánchez Márquez et al. 2007 ; Kusari et al.2009b, 2013 ,2014a).
Our work on analyzing the diversity of endophytic microorganisms inCannabis
plants led to the identification of a plethora of fungal and bacterial endophytes
(Kusari et al. 2013 ,2014a,b). A total of 30 endophytic fungal isolates and 13
endophytic bacterial isolates were identified and characterized. Given that we were
unable to prospect wild populations ofC. sativaplants, this low incidence of
endophytes was expected. The majority of fungal isolates were harbored in apical or
lateral buds (16 isolates), followed by leaves (8 isolates), and finally twigs
(6 isolates). In general, phylum Ascomycota comprises of more than 3000 genera of
mostly plant pathogens (Berbee 2001 ; Heckman et al. 2001 ; Mueller and Schmit
2007 ). Interestingly, majority of endophytes discovered so far belong to phylum
Ascomycota. In our work, all the fungal isolates belonged to Ascomycota, whereby
Penicilliumwas found as the major genus followed byChaetomium,Aspergillius
and Paecilomyces. 16S rRNA identification of bacterial isolates revealed the
presence of Bacillus as the major genus followed by Brevibacillus and
non-pathogenic strains ofMycobacterium. Our results were comparable to another
systemic study ofCannabisendophytes by Gautam et al. ( 2013 ), which showed
host plant colonization by fungal endophytes belonging to 8 different genera,
whereby Aspergilluswas recorded as the most dominant genus followed by
Penicillium, Phoma, Rhizopus, Colletotrichum, Cladosporium and Curvularia.
Similarly,Alternariawas the predominant genus of fungal endophytes isolated
fromCannabisplants prospected from the Western Himalayas (Qadri et al. 2013 ).
Bacterial endophytic isolates from roots and rhizosphere ofCannabisplants from
the wild revealed the presence of diverse genera like Acinetobacter,
Chryseobacterium,Enterobacter,MicrobacteriumandPseudomonas(Afzal et al.
2015 ). All the endophytes were characterized by established molecular methods
based on ITS (ITS1, intervening 5.8S, and ITS2) and 16S rRNA analyses. Apart
from molecular analysis, microscopic and macroscopic identification of endophytes
were also performed to ensure efficacy of isolation procedures. Another important
aspect of our work was to evaluate the fungal endophytic diversity ofCannabis
plants. Quantification and analyses of endophytic biodiversity was performed
employing various approaches including calculation of Menhinick’s index
(Whittaker 1977 ) for evaluation of species richness, and Camargo’s index
(Camargo 1992 ) for extrication of fungal dominance. Further, Fisher’s log series
index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and
Margalef’s richness were calculated for the comprehensive evaluation of the fungal
diversity (Fisher et al. 1943 ; Simpson 1949 ; Margalef 1958 ; Kusari et al. 2013 ).
Overall, the biodiversity was not too high, as expected because the host plants could
not be procured from the natural populations. The apical or lateral buds showed
high species richness, whereas the tissue specific fungal dominance was higher in
twigs. The most dominant species wasPenicillium copticola, belonging to phylum
Ascomycota. The certainty of fungal consistency and taxonomic richness was rel-
atively higher in twigs than in leaves or buds. Nevertheless, the diversity of fungal
422 P. Kusari et al.