scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Pathogenic and Molecular Variability of Aspergillus niger Isolates Causing Collar Rot Disease in Groundnut

28 Feb 2018-International Journal of Pure & Applied Bioscience (Vital Biotech)-Vol. 6, Iss: 1, pp 840-848
TL;DR: Pathogenic variability studies of 26 isolates of collar rot pathogen (Aspergillus niger) of groundnut collected from different districts of Telangana and Andhra Pradesh states were conducted under greenhouse conditions by using two groundnut cultivars TMV-2 and JL-24.
Abstract: Pathogenic variability studies of 26 isolates of collar rot pathogen (Aspergillus niger) of groundnut collected from different districts of Telangana and Andhra Pradesh states were conducted under greenhouse conditions by using two groundnut cultivars TMV-2 and JL-24. The mean seedling mortality ranged from 29.7 per cent (MBNRAn-1 (Palkapally)) to 94.4 per cent (ATPAn-1 (Jogannapet)). All the isolates of A. niger showed differential reaction with regard to seedling mortality on JL-24 and TMV-2 cultivars. Similarly genetic relatedness among eight virulent isolates (two isolates from each district) of A. niger from groundnut was assessed by using RAPD analysis. The similarity index values among the A. niger isolates varied from 0.571 (between WGL An-2 and CHT An-3) to 0.229 (between MBNR An-3 and WGL An-2).

Summary (2 min read)

INTRODUCTION

  • Groundnut (Arachis hypogaea L.) is an important food legume grown in Asia and Sub Saharan Africa.
  • Major groundnut growing states of India include Andhra Pradesh, Telangana, Gujarat, Karnataka and Tamil Nadu.
  • Therefore, the present study was taken up to identify the pathogenic and molecular variability among the isolates of A. niger collected from different areas of Telangana and Andhra Pradesh states.

MATERIAL AND METHODS

  • To identify the most effective and virulent isolate of A. niger, the susceptible cultivars TMV-2 and JL 24 were inoculated separately with each fungal isolate by soil inoculation technique 12 under greenhouse conditions.
  • Sorghum grains were pre-soaked in water overnight.
  • The pots were inoculated with A. niger pathogen 3 days prior to sowing and sprinkled with water.
  • The experiment was conducted in Randomized Complete Block Design (RCBD).
  • Standard protocols were used for the isolation of DNA and RAPD analysis 8 .

Isolation of genomic DNA

  • Mycelium (100±150 mg) of respective isolates (A. niger) were crushed using liquid nitrogen into a fine homogenate.
  • Then the samples were incubated in a water bath (Gallen Ramp, England) at 65° C for 30 minutes and centrifuged at 12000 rpm for 10 minutes in a microcentifuge (Eppendorf Centrifuge 5415 D).
  • The supernatant was transferred into fresh tube and to that, equal volume of extraction buffer (phenol: chloroform: isoamyl alcohol, 25:24:1, v:v:v) was added and stirred gently on a vortex mixture until an emulsion had formed.
  • The contents were later mixed by tilting the tubes gently and the tubes were kept at -20 ° C for Copyright © Jan.-Feb., 2018; IJPAB 842 30 minutes to allow the DNA to precipitate.
  • The DNA concentration was estimated by electrophoresis in a 0±8% agarose gel, staining with ethidium bromide (10 lg ml− 1 ) for 30 min and the staining intensity was compared with Lambda DNA markers under UV illumination.

Measurement of DNA Concentration

  • The DNA from all isolates produced clear sharp bands in one per cent agarose gel indicating the good quality of DNA.
  • The DNA was quantified by comparing with the 1 kb size marker (Genei, Bangalore) and by spectrophotometer (Nanodrop ND1000).

RAPD Profiles through Polymerized Chain Reaction (Pcr)

  • Thirteen oligonucleotide primers (Macrogen Inc. Oligo, Seoul) were screened for generating polymorphism among the isolates under the study.
  • The experiment was repeated thrice and results were reproducible.
  • The Oligonucleotide primer sequences used in RAPD technique are given below: PCR amplifications were carried out in 0.2 mL eppendorf tubes with 25 μL reaction mixture which consists of 2.5 μL of 10x Amplified PCR products were subjected to 1.5 per cent agarose gel electrophoresis with 1.0 x TBE as running buffer.
  • The DNA banding profiles were documented in the gel documentation system (Alpha Innotech) and compared with 1 kb DNA ladder (Genei, Bangalore).

Scoring and Data Analysis

  • Each amplified band was considered as RAPD marker and recorded for all samples.
  • Data was entered using a matrix in which all observed bands or characters were listed.
  • The data matrix thus generated (0, 1 for absence and presence of the bands) was used to calculate Jaccard’s similarity coefficient for each pair wise comparison.
  • The similarity coefficients were subjected to Unweighted Pair-Group Method on Arithmetic Average cluster analysis to group the isolates based on their overall similarities.
  • A similarity matrix was generated from the binary data using DICE similarity co-efficient in NTSYs pc package 14 .

RESULTS AND DISCUSSION

  • Pathogenicity of 26 isolates of A. niger collected from different locations were tested on susceptible groundnut cultivars JL 24 and TMV-2 under greenhouse conditions and the data presented in the Table 1.
  • Similarly with Mahaboobnagar isolates the seedling mortality of 49.1 per cent was recorded on JL-24 and 47 per cent on TMV-2 seedling mortality.
  • The isolate ATPAn-1 was highly virulent among all the A. niger isolates showing an average of 94.4 per cent mortality in both cultivars JL-24 and TMV-2 and the isolate which showed highest per cent mortality was used for further studies .

R151, UBC90, GLB-12, GLB-15, GLK-08,

  • A total of 653 reproducible bands were generated with 10 primers (Plate 1 and 2).
  • In each group or sub group isolates from different locations were present indicating high genetic diversity.
  • Even though the isolates CHTAn-3 and WGLAn-2 were isolated from different geographical regions but they showed highest genetic similarity which may be due to the migration of isolates from one place to another.
  • Similar type of variations with the A. niger isolates also observed by several researchers 13&6 .

CONCLUSIONS

  • Pathogenicity studies with 26 isolates of A. niger was tested on two susceptible groundnut cultivars JL-24 and TMV-2 under greenhouse conditions.
  • The mean seedling mortality was highest (94.4%) with the isolate ATPAn-1 whereas, the lowest mean seedling mortality (29.7%) observed with the isolate MBNRAn-1 .
  • Variation in relation to seedling mortality was observed between the isolates collected from different localities and also between isolate and cultivars.
  • Molecular variability of eight virulent isolates of A. niger with RAPD marker indicated that considerable genetic variation was observed among the isolates.
  • The isolates MBNR An-1 and MBNRAn-3 were grouped into different clusters which clearly indicate the highest level of genetic similarity between the isolates collected from same district.

Did you find this useful? Give us your feedback

Content maybe subject to copyright    Report

Divya Rani et al Int. J. Pure App. Biosci. 6 (1): 840-848 (2018) ISSN: 2320 7051
Copyright © Jan.-Feb., 2018; IJPAB 840
Pathogenic and Molecular Variability of Aspergillus niger Isolates Causing
Collar Rot Disease in Groundnut
V. Divya Rani
1*
, Hari Sudini
2
, P. Narayan Reddy
3
, U. Naga Mangala
4
and
K. Vijay Krishna Kumar
5
1
Professor Jayashankar Telangana State Agricultural University, Hyderabad, Telangana, India
2
International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India
3
Professor Jayashankar Telangana State Agricultural University, Hyderabad, Telangana, India
4
International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India
5
Acharya N G Ranga Agricultural University, Andhra Pradesh, India
*Corresponding Author E-mail: divyavallapu@gmai.com
Received: 26.08.2017 | Revised: 30.09.2017 | Accepted: 7.10.2017
INTRODUCTION
Groundnut (Arachis hypogaea L.) is an
important food legume grown in Asia and Sub
Saharan Africa. It is also an important source
of oil for majority of human population
worldwide and is a rich source of protein for
human and animal consumption.
Approximately, groundnut kernels contain 48-
50% of edible oil and 26-28 % protein, along
with rich dietary fibre, minerals and
vitamins
11
. Major groundnut growing states of
India include Andhra Pradesh, Telangana,
Gujarat, Karnataka and Tamil Nadu. Of these,
Andhra Pradesh, Telangana and Gujarat
contribute to more than half the crop area in
the country
3
. Groundnut cultivation in India as
a rainfed crop is often subjected to significant
yield losses annually due to biotic and abiotic
stresses are the major limiting factors for
attaining high productivity in India. Of various
biotic stresses, soilborne diseases account for
reduced pod yields.
Available online at www.ijpab.com
DOI: http://dx.doi.org/10.18782/2320-7051.5534
ISSN: 2320 7051
Int. J. Pure App. Biosci. 6 (1): 840-848 (2018)
ABSTRACT
Pathogenic variability studies of 26 isolates of collar rot pathogen (Aspergillus niger) of
groundnut collected from different districts of Telangana and Andhra Pradesh states were
conducted under greenhouse conditions by using two groundnut cultivars TMV-2 and JL-24. The
mean seedling mortality ranged from 29.7 per cent (MBNRAn-1 (Palkapally)) to 94.4 per cent
(ATPAn-1 (Jogannapet)). All the isolates of A. niger showed differential reaction with regard to
seedling mortality on JL-24 and TMV-2 cultivars. Similarly genetic relatedness among eight
virulent isolates (two isolates from each district) of A. niger from groundnut was assessed by
using RAPD analysis. The similarity index values among the A. niger isolates varied from 0.571
(between WGL An-2 and CHT An-3) to 0.229 (between MBNR An-3 and WGL An-2).
Key words:
Groundnut, Collar rot, Aspergillus niger, Pathogenic variability, Molecular
variability
Research Article
Cite this article: Divya Rani, V., Sudini, H., Reddy, P.N., Mangala, U.N.
and Kumar, K.V.K., Pathogenic
and Molecular Variability of Aspergillus niger Isolates Causing Collar Rot Disease in Groundnut, Int. J.
Pure App. Biosci. 6(1): 840-848 (2018). doi: http://dx.doi.org/10.18782/2320-7051.5534

Divya Rani et al Int. J. Pure App. Biosci. 6 (1): 840-848 (2018) ISSN: 2320 7051
Copyright © Jan.-Feb., 2018; IJPAB 841
Among soilborne diseases collar rot
(Aspergillus niger Van Tieghem), causing
major havoc in all crop growing areas. Collar
rot disease is commonly manifested as a pre-
and post-emergence damping-off of the
affected seedlings. Occasionally, collar rot can
continue up to crop harvesting stage resulting
in damage to the seed
4
. The fungus also
produces heat canker symptoms
2.
Though, the
annual yield losses due to collar rot alone are
approximately 5 %, the disease has a potential
to damage the crop with 40% losses
1
. For
successful implementation of management
practices against soilborne diseases,
knowledge on pathogen distribution and their
diversity in major crop growing areas is
essential. This will enable to devise location
specific management practices to curb these
soil borne diseases hitherto difficult to manage
especially under high disease pressure.
Therefore, the present study was taken up to
identify the pathogenic and molecular
variability among the isolates of A. niger
collected from different areas of Telangana
and Andhra Pradesh states.
MATERIAL AND METHODS
To identify the most effective and virulent
isolate of A. niger, the susceptible cultivars
TMV-2 and JL 24 were inoculated separately
with each fungal isolate by soil inoculation
technique
12
under greenhouse conditions. The
test pathogen was mass multiplied on sorghum
grains
5
. Sorghum grains were pre-soaked in
water overnight. Later, the excess water was
removed and the soaked grains were
transferred into 1000 ml flasks @ 400 g and
autoclaved at 15 lb psi (121.6
o
C) for 20 min.
The flasks were allowed to cool at room
temperature and inoculated with 5 mm discs of
actively growing 3-4 day old culture of A.
niger. Seven discs per flask were added and
the flasks were later incubated for 10 days at
28 ± 2
o
C.
Soil inoculation technique was
followed for inoculation of the test isolates. To
each pot (5” diameter with sterilized soil), 40 g
of inoculum multiplied on sorghum grains was
added and mixed with the soil to a depth of 5
cm and covered with the soil. The pots were
inoculated with A. niger pathogen 3 days prior
to sowing and sprinkled with water. Later, five
groundnut seeds were sown in each pot. Three
replications were maintained for each isolate.
Per cent seedling mortality was calculated at
30 DAS. The experiment was conducted in
Randomized Complete Block Design (RCBD).
The molecular variability of A. niger
were studied using Random Amplified
Polymorphic DNA (RAPD). Two virulent
isolates from each district were selected for
each pathogen to study the molecular
variability. A total of 13 primers were used.
Standard protocols were used for the isolation
of DNA and RAPD analysis
8
.
Isolation of genomic DNA
Mycelium (100±150 mg) of respective isolates
(A. niger) were crushed using liquid nitrogen
into a fine homogenate. Lysis buffer (200 mM
Tris pH 8; 500 mM NaCl; 100 mM EDTA pH
8 and .0.5% SDS;) was added (800 µl) to the
tube containing homogenate and mixed
thoroughly. Then the samples were incubated
in a water bath (Gallen Ramp, England) at 65°
C for 30 minutes and centrifuged at 12000
rpm for 10 minutes in a microcentifuge
(Eppendorf Centrifuge 5415 D). The
supernatant was transferred into fresh tube and
to that, equal volume of extraction buffer
(phenol: chloroform: isoamyl alcohol, 25:24:1,
v:v:v) was added and stirred gently on a vortex
mixture until an emulsion had formed. The
contents were later centrifuged at 12000 rpm
for 10 min in a micro-centrifuge at room
temperature. An aliquot of the upper aqueous
layer was collected, mixed with an equal
volume of extraction buffer, and recentrifuged.
The supernatant was transferred into new
tubes, mixed with an equal volume of
chloroform and isoamyl alcohol (24:1), and
recentrifuged. The upper aqueous layer was
collected into eppendorf and to this 3µl of
RNase A (10 mg ml
-1
) was added and the
mixture was incubated at 37 °C for 30 min.
Finally 2/3
rd
volume of ice cold isopropanol
was added to the eppendorf tubes. The
contents were later mixed by tilting the tubes
gently and the tubes were kept at -20 ° C for

Divya Rani et al Int. J. Pure App. Biosci. 6 (1): 840-848 (2018) ISSN: 2320 7051
Copyright © Jan.-Feb., 2018; IJPAB 842
30 minutes to allow the DNA to precipitate.
Later, the contents were spinned at 12000 rpm
for 10 minutes in a Centrifuge and the DNA
pellet was washed with 70 % ice cold ethanol
two times to remove the salts and other
impurities and the tubes were air dried for
10±15 min, suspended in 40 µl of TE buffer
(10 mm Tris±HCl (pH 8±0) 1 mm EDTA).
The DNA concentration was estimated by
electrophoresis in a 0±8% agarose gel, staining
with ethidium bromide (10 lg ml−
1
) for 30 min
and the staining intensity was compared with
Lambda DNA markers under UV illumination.
Measurement of DNA Concentration
The quality and quantity of DNA were
analyzed by running 1 μL of each sample
mixed with 1 μL of 10x loading dye
(Bromophenol blue) in 1% agarose gel. The
DNA from all isolates produced clear sharp
bands in one per cent agarose gel indicating
the good quality of DNA. The DNA was
quantified by comparing with the 1 kb size
marker (Genei, Bangalore) and by
spectrophotometer (Nanodrop ND1000).
RAPD Profiles through Polymerized Chain
Reaction (Pcr)
Primer sequences (5’-3’) for Aspergillus niger
S. No
Primers
1
OPA 2
2
OPA 4
3
OPA10
4
OPA 20
5
R108
6
R151
7
UBC90
8
GLB-12
9
GLB-15
10
GLK-08
11
GLL-04
12
GLL-05
13
GLL-12
Thirteen oligonucleotide primers (Macrogen
Inc. Oligo, Seoul) were screened for
generating polymorphism among the isolates
under the study. The experiment was repeated
thrice and results were reproducible. The
Oligonucleotide primer sequences used in
RAPD technique are given below:
PCR amplifications were carried out
in 0.2 mL eppendorf tubes with 25 μL reaction
mixture which consists of 2.5 μL of 10x Taq
buffer, 2 μL of 25 mM MgCl2, 1 μL of primer
(5 picomolar / μL), 1 μl of 5 mM dNTP mix,
0.5μL of Taq polymerase enzyme (conc. 5 U
μL-1) and 15.5 μL of sterile PCR water
(Genei, Bangalore) and 2.5 μL (40-50 ng) of
DNA sample. Amplification was carried out
by 5 min of initial denaturation at 94°C
followed by 40 cycles of denaturation of 94°C
for 1 min; annealing at 37°C for 1 min;
extension at 72°C for 2 min with final
elongation at 72°C for 5 min. Amplified PCR
products were subjected to 1.5 per cent
agarose gel electrophoresis with 1.0 x TBE as
running buffer. The banding patterns were
visualized under UV trans-illuminator with
ethidium bromide (10 mg mL
-1
) staining. The
DNA banding profiles were documented in the
gel documentation system (Alpha Innotech)
and compared with 1 kb DNA ladder (Genei,
Bangalore).

Divya Rani et al Int. J. Pure App. Biosci. 6 (1): 840-848 (2018) ISSN: 2320 7051
Copyright © Jan.-Feb., 2018; IJPAB 843
Scoring and Data Analysis
Each amplified band was considered as RAPD
marker and recorded for all samples. Data was
entered using a matrix in which all observed
bands or characters were listed. The data
matrix thus generated (0, 1 for absence and
presence of the bands) was used to calculate
Jaccard’s similarity coefficient for each pair
wise comparison. The coefficients were
calculated In Silico
7
. The similarity
coefficients were subjected to Unweighted
Pair-Group Method on Arithmetic Average
(UPGMA) cluster analysis to group the
isolates based on their overall similarities. A
similarity matrix was generated from the
binary data using DICE similarity co-efficient
in NTSYs pc package
14
.
RESULTS AND DISCUSSION
Pathogenicity of 26 isolates of A. niger
collected from different locations were tested
on susceptible groundnut cultivars JL 24 and
TMV-2 under greenhouse conditions and the
data presented in the Table 1. All the isolates
were found more or less effective in inducing
seedling mortality in both the susceptible
cultivars. The mean seedling mortality ranged
from 29.7 per cent (MBNRAn-1 (Palkapally))
to 94.4 per cent (ATPAn-1 (Jogannapet)). The
isolates ATPAn-1 (94.4%), ATPAn-6 (76.4%),
ATPAn-3 and CHTAn-3 (70.8%) did not show
any significant difference in the seedling
mortality. Among the isolates collected from
Anantapur isolate ATPAn-4 showed a seedling
mortality of 42.2 per cent while isolate
ATPAn-1 recorded seedling mortality as high
as 94.4 per cent. Similarly, for isolates from
Mahaboobnagar, the seedling mortalities
varied from 29.7 per cent (MBNRAn-1) to
65.6 per cent (MBNRAn-9). While with
Chittoor isolates, the seedling mortality varied
from 32.8 per cent (CHTAn-5) to 70.8 per cent
(CHTAn-3). Aspergillus niger isolates from
Warangal recorded seedling mortality of 41.7
per cent (WGLAn-1) to 59.7 per cent
(WGLAn-2). Further it was also also observed
that isolate ATPAn-1, the seedling mortality
was 88.9 per cent in the cultivar TMV-2,
whereas, 100 per cent mortality was observed
on JL-24. In TMV-2, the seedling mortality
ranged from 33.3 per cent (MBNRAn-1, 4 and
%) to 88.9 per cent (ATPAn-1) and on JL-24,
it varied from 22.2 per cent (MBNRAn-2) to
100 per cent (ATPAn-1). The average per cent
seedling mortality of Anantapur isolates was
61.2 per cent on JL-24 and 65.9 per cent on
TMV-2. Similarly with Mahaboobnagar
isolates the seedling mortality of 49.1 per cent
was recorded on JL-24 and 47 per cent on
TMV-2 seedling mortality. For Chittoor
isolates, the seedling mortality was 38.4 per
cent on JL-24 and 70.9 per cent on TMV-2.
The per cent seedling mortality of Warangal
isolates was 42.8 per cent on JL-24 and 60.6
per cent on TMV-2. The isolate ATPAn-1 was
highly virulent among all the A. niger isolates
showing an average of 94.4 per cent mortality
in both cultivars JL-24 and TMV-2 and the
isolate which showed highest per cent
mortality was used for further studies
(Figure 1).
The present studies indicate the
pathogenic variability of different isolates of
A. niger collected from different regions. All
the isolates of A. niger showed differential
reaction with regard to seedling mortality on
JL-24 and TMV-2 cultivars. Isolates ATP An-
1, ATP An-3, ATP An-6, MBNR An-3, 5, 6
and 9 were highly virulent on JL-24. While the
rest of the isolates were more virulent on
TMV-2. Isolates from the same district have
shown differential reaction on different
cultivars this may be due to the characteristics
of that particular cultivar or due to the agro
ecological conditions of that locality from
which the isolate was collected and also due to
the variation in the characteristics of the
isolate
15
. Similar type of variations with the A.
niger on groundnut cultivars was also
observed by the earlier workers where the

Divya Rani et al Int. J. Pure App. Biosci. 6 (1): 840-848 (2018) ISSN: 2320 7051
Copyright © Jan.-Feb., 2018; IJPAB 844
isolates (AN1 to AN5) showed marked
differences in the per cent germination, per
cent seed rot and per cent collar rot on
groundnut cultivar AK12-24. The isolate AN1
showed lowest seed germination and highest
collar rot incidence considered to be more
virulent
9
.
Molecular variability among isolates of
Aspergillus niger
Genetic relatedness among eight virulent
isolates (two highly virulent isolates (based on
seedling mortality values) from each district)
of A. niger from groundnut was assessed by
using RAPD analysis. Thirteen primers viz.,
OPA 2, OPA 4, OPA 10, OPA 20, R108,
R151, UBC90, GLB-12, GLB-15, GLK-08,
GLL-04, GLL-05 and GLL-12 were screened
of which 10 primers (OPA 2,OPA 4,OPA10,
OPA 20, R108, R151, UBC90, GLB-12, GLB-
15 and GLK-08) generated reproducible
polymorphism among the eight tested isolates.
A total of 653 reproducible bands were
generated with 10 primers (Plate 1 and 2).
The data presented in the Table 2
indicated that similarity index values among
the A. niger isolates varied from 0.571
(between WGLAn-2 and CHTAn-3) to 0.229
(between MBNRAn-3 and WGLAn-2) (Table
2). The isolates collected from
Mahaboobnagar were highly variable and they
share only 23 % similarity followed by
Warangal 39.5 %, Chittoor 50% and
Anantapur 55.7 %. Genetic similarity between
the A. niger isolates was estimated based on
UPGMA cluster analysis (Figure 2) and
indicated that all the isolates were grouped
into two major clusters with single isolates i.e.,
MBNR An-1 and MBNRAn-3. The first
cluster was further subdivided into four
isolates which was again subdivided into two
sub cluster of two each i.e. ATPAn-1 and
ATPAn-6; CHTAn-3 and WGLAn-2. The
isolates present in second cluster (CHTAn-4
and WGLAn-3) were more diverse when
compared to first one. Highest similarity was
observed between isolates collected from
Chittoor (CHTAn-3) and Warangal (WGL An-
2). Remaining isolates showed considerable
genetic variation with each other.
These findings have clearly shown
that RAPD can genetically differentiate the
isolates at inter specific level. Correlation was
not observed between the similarity index and
the place of collection of isolates and
clustering also was not observed according to
location except ATPAn-1, ATPAn-6. In each
group or sub group isolates from different
locations were present indicating high genetic
diversity. Even though the isolates CHTAn-3
and WGLAn-2 were isolated from different
geographical regions but they showed highest
genetic similarity which may be due to the
migration of isolates from one place to
another. Similar type of variations with the A.
niger isolates also observed by several
researchers
13&6
. Similarly
10
studied the
molecular variability of 17 mutants and a wild
type strain of A. niger using 12 random
primers. All the mutants showed a varying
degree of genetic divergence based on their
amplification profile and maximum number of
amplifications in wild type A. niger strain were
produced by primer K-16. All the mutants
were genetically diverse with wild type strain.
The maximum similarity (78.63%) with wild
type strain was found in G-80-A. On the other
hand, the most closely related mutants were
UV-180-A and UV-180-B with the genetic
similarity of 61.60 %. From our results study
of genetic variation through RAPD primers is
useful in estimating the diversity among the
isolates, between and within same species
collected from the same host. The present
findings indicated that genetic differences
between species of the same genus maintain
genetic diversity within this population
isolated from same host.

Citations
More filters
Journal ArticleDOI
TL;DR: Nguyen et al. as mentioned in this paper identified and characterized Aspergillus niger causing collar rot of groundnut (Arachis hypogaea) from nine different locations in Quang Tri and Thua Thien Hue provinces.
Abstract: Abstract. Nguyen XH, Nguyen TMN, Nguyen DH, Nguyen QC, Cao TT, Pham TTH, Nguyen TTT. 2023. Identification and characterization of Aspergillus niger causing collar rot of groundnut (Arachis hypogaea). Biodiversitas 24: 2556-2562. Groundnut (Arachis hypogaea L.) is an economically important legume crop in Vietnam and many other countries worldwide. Groundnut cultivation is affected by many biotic and abiotic stresses. Among biotic stresses, groundnut is attacked by many fungal, bacterial and viral pathogens. The most harmful fungal diseases are rust, stem rot, collar rot and other soil borne diseases. Collar rot caused by Aspergillus niger is one of the most important disease of groundnut extensive in Vietnam and worldwide. In this study, symptomatic infected plant samples were collected from nine different locations in Quang Tri and Thua Thien Hue provinces, Vietnam Isolated fungal species were identified on the basis of morphological characterization, and molecular level. The pathogenicity of fungal species was confirmed on the basis of Koch's postulates. The result showed that a total of nine fungal strains were isolated from infected groundnut samples. Morphological analysis results showed that all isolates exhibited A. niger characteristics, including black colony, biseriate conidia, hyaline conidiophores and sterigmata. Pathogenicity test showed that all nine A. niger isolates caused collar rot disease in groundnuts. Further molecular identification based on ITS1-4 region nucleotides comparison indicated that selected two isolates, namely QT1 and H17 belonged to A. niger. Further research should be done to find antagonistic microorganisms isolates for the bio-control of A. niger.
References
More filters
Journal ArticleDOI
TL;DR: A rapid, low-cost, and reliable DNA extraction procedure for fungi that can be completed within an hour and is applicable to various filamentous fungi.
Abstract: Nucleic acid detection methods such as PCR have become a common tool for microbial identification and diagnosis. Although PCR amplification can be performed directly for various microbial cultures, for filamentous fungi and yeasts, prior isolation of DNA is often preferred. As the DNA extraction process eliminates many unknown interfering substances present in the biological material, it plays an important role in ensuring consistent test results. Toward this end, considerable efforts have been made to enable improved DNA preparation from fungi (1–3). Many of these methods rely on using a grinder (with or without liquid nitrogen) for initial breaking up of the mycelia. This is a significant handicap when dealing with a large number of samples. Being a clinical diagnostic laboratory, we routinely perform PCR experiments on a variety of fungal pathogens. The availability of a rapid, low-cost, and reliable DNA extraction procedure for fungi not only would reduce the workload considerably but also would decrease the test turnaround time. After experimenting with several DNA purification regimens, we have optimized a rapid, mini-preparation procedure for fungal DNA. This procedure includes the following steps. (i) To a 1.5-ml Eppendorf tube containing 500 μl of lysis buffer (400 mM Tris-HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate), a small lump of mycelia is added by using a sterile toothpick, with which the lump of mycelia is disrupted. The tube is then left at room temperature for 10 min. (ii) After adding 150 μl of potassium acetate (pH 4.8; which is made of 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of distilled water), the tube is vortexed briefly and spun at >10,000 × g for 1 min. (iii) The supernatant is transferred to another 1.5-ml Eppendorf tube and centrifuged again as described above. After transferring the supernatant to a new 1.5-ml Eppendorf tube, an equal volume of isopropyl alcohol is added. The tube is mixed by inversion briefly. (iv) The tube is spun at >10,000 × g for 2 min, and the supernatant is discarded. The resultant DNA pellet is washed in 300 μl of 70% ethanol. After the pellet is spun at 10,000 rpm for 1 min, the supernatant is discarded. The DNA pellet is air dried and dissolved in 50 μl of 1× Tris-EDTA, and 1 μl of the purified DNA is used in 25 to 50 μl of PCR mixture. The whole procedure can be completed within an hour and is applicable to various filamentous fungi. Up to now, we have used the technique to isolate DNA from 150 dermatophytes (including 110 Trichophyton, 30 Microsporum, and 10 Epidermophyton isolates) and 10 Fusarium isolates. In addition, we also succeeded in generating sufficient DNA from yeasts (10 Candida and 10 Cryptococcus isolates) as well as bacteria (180 Staphylococcus and 10 Campylobacter isolates) using this rapid method. The DNA yields from filamentous fungi and bacteria were reasonably high, and a clear DNA band was frequently seen when 10 μl of the 50-μl DNA preparation was run in an agarose gel and stained with ethidium bromide. However, the amount of DNA obtained from Candida and Cryptococcus spp. was considerably lower, and only a very faint DNA band was observed when the DNA preparations were assessed by agarose gel electrophoresis, particularly those from Cryptococcus isolates. Nevertheless, genomic DNA extracted by the procedure, including that from Candida and Cryptococcus spp., has been readily amplified by PCR. It is likely that this procedure could be applied to the examination of many other fungal cultures and, possibly, clinical specimens. It provides a rapid, reliable, and low-cost alternative to the existing DNA purification protocols used in research and clinical laboratories.

421 citations

01 Jan 2008
TL;DR: The contents of the document are technical aspects of groundnut seed production, classes of seed, seed certification standards, monitoring and inspection, crop maintenance, industrial preparation of ready-to-use groundnut seeds, seed physiology and seed systems in West Africa.
Abstract: The contents of the document are (1) groundnut crop (2) classes of seed (3) seed certification standards (4) monitoring and inspection (5) technical aspects of groundnut seed production (6) crop maintenance (7) industrial preparation of ready-to-use groundnut seeds (8) seed physiology (9) and seed systems in West Africa.

43 citations

Journal ArticleDOI
TL;DR: The high levels of genetic variation attributed to parasexuality and/or wind-facilitated gene flow from an as of yet undocumented broader host range of the fungus on other desert vegetation are attributed to A. niger infections of W. mirabilis.
Abstract: Aspergillus niger is an asexual, haploid fungus which infects the seeds of Namibia's national plant, Welwitschia mirabilis, severely affecting plant viability. We used 31 randomly amplified polymorphic DNA markers to assess genetic variation among 89 A. niger isolates collected from three W. mirabilis populations in the Namib Desert. While all isolates belonged to the same vegetative compatibility group, 84% were unique genotypes, and estimates of genotypic evenness and Simpson's index of diversity approached 1.0 in the three populations. Analysis of molecular variance revealed that 78% of the total variation sampled was among isolates from individual W. mirabilis plants. Lower, but significant, amounts of variation detected among isolates from different plants (12%) and different sites (10%) also indicated some site- and plant-level genetic differentiation. Total gene diversity (H(T) = 0.264) was mostly attributable to diversity within populations (H(S) = 0.217); the relatively low level of genetic differentiation among the sites (G(ST) = 0.141) suggests that gene flow is occurring among the three distant sites. Although sexual reproduction has never been observed in this fungus, parasexuality is a well-known phenomenon in laboratory strains. We thus attribute the high levels of genetic variation to parasexuality and/or wind-facilitated gene flow from an as of yet undocumented broader host range of the fungus on other desert vegetation. Given the apparent ease of transmission, high levels of genetic diversity, and potentially broad host range, A. niger infections of W. mirabilis may be extremely difficult to control or prevent.

23 citations

Journal ArticleDOI
TL;DR: Based on collar rot disease incidence, groundnut varieties were screened as: susceptible, moderately susceptible and tolerant and Trichoderma viride 60 (seed treatment) in pot culture showed significant differences in the per cent of disease incidence of collar rot, up to 15 days after sowing (DAS).
Abstract: Antagonistic effect of 12 isolates of 3 Trichoderma strains (T. virens, T. viride, T. harzianum) against the collar rot disease-caus- ing fungus A. niger, was studied in vitro. It was observed that T. viride 60 inhibited maximum (86.2%) growth of test fungus, followed by T. harzianum 2J (80.4%). The five varieties of groundnut grown in normal (T 1 ), sick - A. niger infested soil (T 2 ) and sick + Trichoderma viride 60 (seed treatment) (T 3 ) in pot culture showed significant differences in the per cent of disease incidence of collar rot, up to 15 days after sowing (DAS). The per cent of collar rot disease incidence was higher in the GG-20 (67.4%) variety, followed by moderate in GAUG-10 and GG-13 (46%), and minimum in J-11 and GG-2 (30%) in A. niger infected pot culture, at 15 days after sowing (DAS). Based on collar rot disease incidence, groundnut varieties were screened as: susceptible, moderately susceptible and tolerant. Tricho- derma seed treatment (T 3 ) reduced 51.6% of the disease incidence in susceptible varieties and 58.1% in tolerant varieties, at 15 DAS, under A. niger infection (T 2 ) in pot culture study.

20 citations

Frequently Asked Questions (2)
Q1. What have the authors contributed in "Pathogenic and molecular variability of aspergillus niger isolates causing collar rot disease in groundnut" ?

Rani et al. this paper conducted pathogenic variability studies of 26 isolates of collar rot pathogen ( Aspergillus niger ) of groundnut collected from different districts of Telangana and Andhra Pradesh states were conducted under greenhouse conditions by using two groundnut cultivars TMV-2 and JL-24. 

4 per cent mortality in both cultivars JL-24 and TMV-2 and the isolate which showed highest per cent mortality was used for further studies ( Figure 1 ).