Interaction of Beauveria bassiana (Bals.) Vuill. with Leptocorisa oratorius Fab. (Hemiptera: Alydidae) with special reference to chitinase
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Date
2021
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Abstract
Rice earhead bug, Leptocorisa oratorius Fab. and Leptocorisa acuta Thun.
(Hemiptera: Alydidae) are the most important sucking pests of rice. The insect cause
significant yield losses to the rice crop in the field condition. Therefore, the present
investigation was carried out with a view to study the seasonal incidence of L. oratorius
in relation to the meteorological parameters, their damage potential, biology,
pathogenicity of Beauveria bassiana (Bals.) Vuill. against different stages of
L. oratorius and to characterize the B. bassiana-chitinase that secreted during insect
fungus interaction.
To study the seasonal incidence of L. oratorius, an experiment was conducted at
Instructional Cum Research (ICR) Farm, Assam Agricultural University, Jorhat during
Ahu, 2018 & 2019 and Sali (Kharif), 2018 & 2019. During Ahu, 2018 & 2019, the
population of L. oratorius was first appeared on 2nd week of May i.e. 20th standard
meteorological week (SMW) with an average of 0.33 and 0.30 adult per hill,
respectively. The population was gradually increased with a peak of 2.66 and 1.56 adult
per hill on 24th SMW (2nd & 3rd week of June), respectively. During Sali (Kharif) 2018
& 2019, the population of L. oratorius was first appeared on 41st standard (SMW) with
an average of 0.30 and 0.36 adult per hill, respectively. The population was found to be
highest at 1st week of November i.e. 44th SMW with an average of 1.20 and 1.13 adult
per hill, respectively. The correlation studies between incidences of L. oratorius with
meteorological parameters showed non significant correlation with all the
meteorological parameters during Ahu, 2018 and Sali (Kharif), 2018 and 2019.
However, during Ahu 2019, it showed significant positive correlation with maximum (r
= 0.802) & minimum (r = 0.711) temperature and BSSH (r = 0.631) whereas, significant
negative correlation with morning (r = -0.849) & evening (r = -0.750) relative humidity
and non significant negative correlation with rainfall (r = -0.255). Multiple regression
analysis during Ahu 2019, revealed that only morning relative humidity determined the
incidence of L. oratorius in concert with R2 =0.721 (72.10%) and adjusted R2= 0.691
(69.10 %). The „best fit model‟ Y= 12.82531-0.13115*Mor. RH (%) expressed the
magnitude of the relationship.
The percent grain infestation due to rice earhead bug were recorded as 39.34 and
37.11 during Ahu, 2018 & 2019 and 35.43 & 36.28 during Sali (Kharif), 2018 & 2019,
respectively.
The biology of L. oratorius and L. acuta were studied under laboratory
condition. The preoviposition and oviposition period of L. oratorius were found to be
12.00±7.17 and 11.60±4.35 days whereas, 7.40±3.50 and 10.20±5.71 days were
recorded incase of L. acuta, respectively. A gravid female of L. oratorius laid on an
average 99.20±22.01 number of eggs during her life span whereas, 76.80±21.79 number
of eggs was laid by L. acuta. The incubation period of L. oratorius and L. acuta were 5.80±1.09 and 5.00±1.00 days, respectively. Both the species have five nymphal instars.
The duration of each instar of L. oratorius were 3.80±0.84, 2.20±0.45, 3.60±0.89,
6.80±0.84 and 4.80±0.84 days whereas, 4.40±1.14, 4.80±1.09, 3.40±1.14, 3.80±1.48
and 6.00±1.22 days incase of L. acuta. The total nymphal period and total
developmental period of L. oratorius were found to be 21.20±1.09 and 27.00±1.58 days
whereas, 22.40±1.14 and 27.40±2.02 days were found incase of L. acuta, respectively.
Adult male of L. oratorius lived for 28.60±2.40 days and female lived for 57.60±7.06
days. The longevity of both male and female of L. acuta were 28.40±5.81 and
51.80±6.37 days, respectively.
The morphometric study of L. oratorius revealed that the average length of
eggs, first, second, third, fourth and fifth instar were 1.02±0.06, 1.49±0.06, 2.82±0.05,
5.05±0.06, 10.61±0.09 and 14.03±0.15 mm whereas, width were found as 0.75±0.05,
0.28±0.04, 0.40±0.05, 0.48±0.05, 1.07±0.07 and 1.39±0.06 mm, respectively. The
female L. oratorius was slightly shorter than the male and measured 17.08±0.16 mm in
length and 1.98±0.09 mm in width while the male bug was slightly longer than female
measured 18.01±0.16 mm in length and 2.11±0.11 mm in width. In case of L. acuta the
average length of eggs, first, second, third, fourth and fifth instar were recorded to be
1.27±0.04, 1.39±0.05, 2.41±0.08, 3.75±0.07, 5.46±0.11 and 6.68±0.12 mm whereas,
width were found to be 0.52±0.03, 0.55±0.07, 0.59±0.05, 1.38±0.08, 1.75±0.05 and
2.29±0.04 mm, respectively. The average length and width of male bug were
10.23±0.12 and 2.31±0.04 mm whereas, female bug were 9.85±0.12 and 2.39±0.44 mm,
respectively.
The head length and width of L. oratorius varied as 2.35±0.07 and 0.82±0.07
mm whereas, incase of L. acuta 1.29±0.02 and 0.53±0.02 mm, respectively. Likewise
thorax length, thorax width, abdomen length, abdomen width, distance between two
compound eyes, length of compound eyes, width of compound eyes, antennal length,
stylet length, fore wing length, fore wing width, hind wing length, hind wing width, fore
leg length, middle leg length and hind leg length of L. oratorius were also measured as
3.16±0.15, 2.05±0.07, 12.65±0.13, 2.23±0.14, 1.03±0.13, 0.89±0.10, 0.73±0.08,
14.74±0.12, 5.60±0.05, 12.20±0.19, 2.00±0.14, 8.51±0.16, 2.02±0.09, 9.07±0.25,
9.85±0.27 and 14.22±0.20 mm whereas, 1.60±0.02, 1.94±0.04, 6.00±0.07, 2.61±0.05,
1.8±0.08, 0.41±0.03, 0.29±0.02, 5.20±0.04, 4.85±0.06, 6.38±0.07, 1.87±0.08,
4.64±0.07, 1.79±0.05, 3.43±0.16, 4.11±0.21 and 5.37±0.16 mm were found incase of
L. acuta, respectively.
The pathogenicity test of B. bassiana against different stages of L. oratorius was
carried out under laboratory condition and found to be effective against all the stages of
this pest. The B. bassiana treated eggs showed 72.00±3.74 percent egg hatchability as
compared to the untreated control (100±0.00). The mean percent mortality of first,
second, third, fourth, fifth instar and adult were recorded as 84.00, 80.00, 66.00, 70.00
and 62.00 and 58.00, respectively at 9 days after treatment (DAT). However, significant differences (P ≤ 0.05) were found among the mean percent mortality of nymphal and
adult stages at different DAT.
Infection process of B. bassiana in insects is mainly triggered by the chitinase
which is secreted during insect fungus interaction. Therefore, an experiment was
conducted in the Department of Agricultural Biotechnology, Assam Agricultural
University, Jorhat during the period 2016-2021 to optimize the growing condition of B.
bassiana for chitinase production, reaction condition for chitinase activity assay and to
purify the enzyme. For the production of B. bassiana-chitinase, different growth
parameters viz., growth period, media composition (peptone concentrations, colloidal
chitin concentrations and ferrous sulphate concentrations), media-pH and growth
temperature were optimized. Along with the different growth parameters, reaction
parameters viz., temperature and pH were also optimized for the estimation of B.
bassiana–chitinase activity. The optimum incubation time required for chitinase
production by B. bassiana was found to be 5 days, peptone concentration as 0.07%,
colloidal chitin concentration as 0.4% and ferrous sulphate (FeSO4.7H2O) concentration
as 0.015%, media pH as 5.0 and growth temperature as 28°C, whereas, for B. bassianachitinase
activity the optimum reaction temperature and reaction pH were found to be
37°C and 5.0, respectively.
Chitinase was purified by ammonium sulphate precipitation method by using
different salt fractions, 55%, 65%, 75%, 85% and 95%. Chitinase activity and protein
were estimated in crude enzyme as well as in its different salt fractions after proper
dialysis. From the estimated chitinase activity and protein, the specific activity, fold
purification and yield were calculated. Among the different fractions, with 75%
ammonium salt saturation showed the best result as it purified the enzyme to the
maximum fold of 2.68 with a yield of 14.34% and specific activity of 2.60 U/mg. The
purity of the enzyme was examined by SDS-PAGE and determined the molecular
weight at the highest purified fraction i.e. 75% ammonium salt saturation. The
molecular weight of purified chitinase was estimated to be around 60 kDa.