Mycorrhizal the Department of Biotechnology, Sarguja University, Ambikapur,

Mycorrhizal
culture and preparation of inoculums

Spores of arbuscular mycorrhizal fungi (AMF) Glomus mosseae were collected from CMCC (Centre for Mycorrhizal
Culture Collection), The Energy and Resources Institute (TERI) New Delhi India.
To prepare a culture of G. mosseae, trap culture experiments were
conducted in the pot at the Department of Biotechnology, Sarguja University,
Ambikapur, Chhattisgarh, India using autoclaved (120?C, 0.2 MPa, 20 min) sand
and commercial peat soil  (W:W = 1:3) as
the culture medium and wheat as the host plant. After the successful trapping
process, the mixtures containing G.
mosseae spores, mycelium, sandy soil and mycorrhizal corm fragments were
used as the inoculums. Every 100 g of the prepared inoculums contained spores
was mycorrhizal (M) and mixed with soil for the field experiment in farmers field
Bhittikala, Ambikapur, Chhattisgarh, India.

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Plant
material and growth conditions

A field experiment was conducted to assess the impact of G. mosseae on three varieties of mung bean
during Ravi season 2017. Mung bean experimental seeds MLT-11, MLT-12 and IVT-29
were collected from Rajmohini Devi Agriculture College and Research Station,
Ambikapur, Chhattisgarh, India. Healthy seeds were selected and surface
sterilized using fungicide Carbendazim then the seeds were sown in field
conditions, for the field experiment following standard agricultural practices
for mung bean, 5 cm distance between plant to plant and 25 cm between row to
row was maintained. The experiment was performed in non-mycorrhizal (NM) group
(NG) and mycorrhizal (M) group for all three varieties. For the mycorrhizal (M)
group (IG), 100 g of prepared inoculums were thoroughly mixed with the soil,
while the same amount of sterilized inoculums was mixed with non-mycorrhizal
(NM) group (NG).

Evaluation
of fungal root colonization

Mung bean plants were harvested after 4-6 months of growth, leaves,
shoots and roots were sampled separately. Sub-samples of fresh nutritive roots
were taken to assess mycorrhizal colonization. Rinsed root samples were cleaned
with 10% KOH at 90?C for 60 min and soaked with 1% HCl for 5 min, and then
stained with lactophenol cotton blue (LPCB) (Phillips & Hayman, 1970). The
proportion of the length of each root segment, which contained the any of the
endophytic elements – hyphae, coils, vesicles and arbuscules – was taken as
evidence of mycorrhization. The percentage of the root length with mycorrhiza
endophytes in the samples were calculated from the frequency distribution.
Percentage root colonization (PRC) was determined as the proportion of the
total number of root segments with hyphae, coils, vesicles and arbuscules by
use of the same root segments as in the previous measurements.

Plant
growth measurement

Plant and root height of all five mung bean varieties were measured
with a tape line and recorded in centimeter after harvest. Remaining roots and
other parts of the plant were rinsed with deionized water three times. Fresh
weight of total leaves, shoots, roots and total plant biomass were measured and
then dried at 70?C in an oven for 48 h to attain constant weight. The
percentage water content of remaining roots and total fresh root weight were
used to estimate total root dry weights in grams. For each treatment group,
three separate plants were selected for analysis.

Chlorophyll
and carotenoid content

Chlorophyll and carotenoid contents of the plants were measured as
per the method reported by Lichtenthaler (1987). In this method, 0.2 g fresh
texture of the leaf was weighed and then grounds in mortar pestle with 80 % acetone.
Then 5 ml acetone was added to it and solution volume was reached to 15 ml.
Three ml of this solution was poured in a cuvette and its absorption intensity
was studied at 470, 663, 647 nm by Spectrophotometer (Make……………………..). For regulating
spectrophotometer, 80 % acetone was used as a control. Pigment density was
determined in terms of mg/g fresh weight of the plant essence.