Chili plants have been
known to possess endophytic fungi within them (1). These endophytic fungi are
the organisms residing in the internal plant tissues without causing any overt symptoms
(Stone JK et al., 2000). These
symptomless organisms offer a wide array of roles in plant protection, plant
resistance to pathogens and biotic / abiotic stresses (Pimentel IC et al.,2006, Procopio REL et al.,2009), secretes bioactive
antimicrobial compounds such as alkaloids, peptides, steroids and phenol (Dandu
A et al.,2013). However,
methodological limitations have limited the exploration of these endophytic
groups and the major challenge lies in the isolation of these organisms owing
to the fact that endophytes are internal to plant structures.
The study of endophytes
is generally regarded as a method-dependent process. Methods involving surface
sterilization and plate culture alone are not a reliable isolation method.
Moreover, these are slow, time consuming, lack sensitivity and specificity, labour
intensive, difficult to interpret and most importantly numerous endophytes may
remain unisolated(Stroble G et al.,2005).
So, a prior microscopic visualization using differential staining of the
various plant parts followed by its plate culture could prove effective to
minimise its limitations.
Plant parts like
epidermal peel from leaf sheath, pith scrapings and seeds have been examined
using various stains (Xu L et al.,2008). Lacto Phenol method can stain the fungi blue
aiding in easier visualization and examination (Rozin P et al.,1980) whereas Cotton blue stains the protoplasm of the
fungus leaving out the cell walls unstained thereby leaving the septa of spores
and mycelium distinctly visible (Perry L et
al.,2007). For the staining of mycelium, Lactophenol trypan blue (Govindappa
M et al.,2005) and aniline blue (Bosland
PW et al.,1999) are employed where
trypan blue stains hyphae better. A general histological stain, Gentian violet,
which is a simple and clear cut stain does not provide differential staining (Perry
L et al.,2007). For detection of endophytic
fungi in plant tissues (including seeds), Rose Bengal stain offers better
visualization, comparatively faster (30-60 sec) and safer than trypan blue (3-5
min) (Xu L et al.,2008). Apart from these, Toluidine blue O, Safranin &
fast green staining, Pianese III B Stain, KOH Aniline blue are used to localise
endophytic fungi within the leaves, Chlorazole Black E stains within the roots
and Rhodamine B / Methyl green method within the plant woods (12).
Therefore the objective
of this study was to a) compare the two stains namely lactophenol trypan blue
and rose Bengal stain for their rapid and effective staining of small amounts
of endophytic fungus within the leaves of four chili varieties of Assam and b)
to analyse and correlate the results of endophytic fungal structures observed
in the staining method with the results of endophytic fungal structures grown
on the media plate. A staining solution of Rose Bengal and Lactophenol trypan
blue were developed and tested in leaf sheath of four different chili varieties
of Assam (Capsicum annum L., Capsicum chinense Jacq., Capsicum assamicum J. Pur – L.
Singh and Capsicum frutescens L.).
Fresh and uninfected leaf samples were used in this study.
A standard solution of
Rose Bengal was prepared: 0.5% rose Bengal dissolved in 5% aqueous ethyl
alcohol. The shelf life of the standard solution is 5-6 months. Firstly, the
leaf was rinsed with distilled water and dried. Leaf sheaths of the selected varieties
was opened and a thin layer on the inside was peeled adaxially. Three to four
peeled sections were placed on a glass slide, and one to two drops of standard
rose Bengal solution was applied to the samples. Staining time was usually
30-60 sec, but could be as short as 15 seconds in well peeled samples. Samples
were covered with cover slip and excess stain was then drawn off (Xu L et al.,2008).
All preparations/slides were examined under Bright Field microscope at 10 X and
40 X magnification.
contained lactic acid, glycerol, liquefied phenol and water (1:1:1:5).
Epidermal tissue was peeled from the leaf sheath and placed on a glass slide.
Three to four drops of lactophenol – 0.1% trypan blue solution was added and
kept for 3-5 minutes. The slide was gently heated over flame and 1-2 drops of
water was added. The excess stain was removed and a cover glass was placed over
the stained samples (Xu L et al.,2008). All preparations/slides
were examined under Bright Field microscope at 10 X and 40 X magnification.
To validate the
probable fungal endophytes, the same leaf tissue was cultured on Potato
Dextrose Agar (PDA) plates after surface sterilisation. The fresh plant leaves
were surface sterilized with distilled water for 1 minute and then washed in
70% ethanol for 1 min and then transferred to 0.5% NaOCl solution for 1 minute
and again to 70% ethanol solution for 1 minute and lastly, in distilled water
for 1 minute. The parts were taken out and then allowed it to dry (Perini, 1992; Werner et al, 1997). These were
carefully placed on Potato Dextrose Agar (PDA) petriplates to isolate the
endophytic fungus and incubated at 28 degree Celsius for 72 hours (Zinniel et al, 2002).
The endophytic fungal
development in different chili varieties by the penetrating hyphae was observed
using bright field microscope. Hyphae were the principal structures observed.
Lactophenol trypan blue stained blue and rose Bengal cotton blue stained red in
contrast with the light green of the plant leaves. Our results showed that both
lactophenol trypan blue staining and rose Bengal staining were quick and
effective method for staining endophytic fungi in plant tissues. Two types of
fungal spores were identified by using both the stains. Spherical shaped spores
and irregular rod shaped spores were principally seen in the various chili
Out of these four chili
varieties, three varieties namely C.assamicum,C.annuum and C.frutescens showed the presence of both spherical shaped spores
and irregular rod shaped spores. C.chinense
exhibited the presence of only one type of spore i.e spherical shaped spores.
Among the two tested
stains, spherical shaped spores were distinctly stained red by using Rose
Bengal Stain whereas, by using Lactophenol-Trypan Blue stain, spherical spores
were not stained uniformly. Irregular rod shaped spores were distinctly stained
with both the dyes but Lactophenol-Trypan blue dye was found to be more
suitable for staining these spores.
The plate culture
methods revealed correlated results with the microscopic examination for fungal
endophytes. In the culture dependent method, C.assamicum showed the presence of both white mat fungal growth and
light brown slimy layer, which clearly correlates to the irregular rod shaped
and spherical shaped spores respectively seen in the microscopic staining
method. C.annuum also showed
interesting results that revealed the presence of both white mat and green mat
type and light brown slimy layer as already seen as irregular rod shaped and
spherical shaped spores respectively in the microscopic staining method. In
addition, C. frutescens also
exhibited the presence of both white mat and light yellow slimy layer as
observed in staining method. In contrast to these three varieties ,C.chinense which revealed only the
presence of spherical shaped spores showed only light brown slimy layer, which
totally correlated the two methods, staining method and culture dependent
Figure 1 :