CELLULAR DISEASES PROGRESSION STUDY OF ASPERGILLUS …

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Shilpashree et al. World Journal of Pharmacy and Pharmaceutical Sciences
CELLULAR DISEASES PROGRESSION STUDY OF ASPERGILLUS
FLAVUS AND FUSARIUM SOLANI INTERACTION WITH CICER
ARIETINUM L. HOST
Department of Studies in Botany, University of Mysore, Manasagangotri, Mysuru-
570 006, Karnataka, India.
ABSTRACT
Chickpea being a rich source of protein is prone to damage by insects,
pests and diseases. Anatomical studies were carried out in a timecourse
manner to elucidate the infection process, Aspergillus flavus and
Fusarium solani are not just common seed borne fungi but they have
the potential to become a severe pathogen especially the Aspergillus
sps. It was found that F. solani produces a characteristic brown spots
on the leaves of the chickpea varieties which ultimately lead to wilting,
A special characteristic infection was produced by the Fusarium solani
on the KAK 2 variety where the infection was only to the midrib and
the lateral restricted veins and not seen in the Mesophyll tissue. Even
though it attacks the vascular system, it does not induce wilting of the
leaves. Comparing the progression of infection, wilting period and the
number of leaves or plant wilted in a given time, it was found that JG 11 was less prone to
infection compared to KAK 2 followed by A1 which was very much prone to infection. Thus,
the inoculation of the above mentioned varieties with A. flavus resulted in similar results,
with varied symptoms and levels of severity.
KEYWORDS: Fusarium solani Aspergillus sps. KAK 2 variety JG 11 and A1.
INTRODUCTION
Chickpea [Cicer arietinum L.] is a legume belongs to genus Cicer, tribe Cicereae, family
Fabaceae. It is the third most important food legume grown in 11 million hectares with 8
million ton production. It is commonly called as „Bengal gram in India. Chickpeas are a type
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
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Volume 5, Issue 8, 1571-1585 Research Article ISSN 2278 – 4357
*Corresponding Author
DOI: 10.20959/wjpps20168-7468
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of pulse, with one seedpod containing two or three peas. India occupies the first position in
the world in terms of chickpea area (6.93 m.ha.) and production (5.60 m.t.) during 2005-06
accounting for nearly 31 per cent and 40 per cent of total pulses area and production
respectively, with a productivity of 808 kg/ha. Karnataka ranks fifth in the cultivation of
chickpea with an area of 4.49 lakh ha, 2.08 lakh tonnes of production and 464 kg/ha of
productivity. Generally, there are two main kinds of chickpea: 1. Microsperma (Desi) which
has small, darker seeds, angular in shape and a rough coat, cultivated mostly in the Indian
subcontinent, Ethiopia, Mexico and Iran. 2. Macrosperma (Kabuli) which has lighter
coloured, larger seeds and a smoother coat mainly grown in Southern Europe, Northern
Africa, Afghanistan, Pakistan and Chile also introduced during the 18th century to the Indian
subcontinent.
It is a self pollinated crop with a diploid chromosome number of 16 (Ahmad and Hymowitz,
1993). The best soils for chickpea growth are deep loams or silt clay loams devoid of soluble
salts (Moolani and Chandra, 1970). The maximum nutrient availability from the soil is at a
pH range of 5.7 to 7"2.
Sprouted Bengal gram is reported to possess higher antioxidant potential along with
inhibitory potential against carbohydrate digestive enzymes relevant to type-2 diabetes
(Pittaway et al., 2008).
More than 50 pathogens of chickpea are identified in the world (Nene et al., 1996) but a few
of them have the potential to devastate the crop.
Chickpea production is severely curtailed by Fusarium wilt caused by F. oxysporum
(Schlechtend.:Fr.) f. sp. ciceris (Padwick) in most chickpea growing areas of the world (Jalali
and Chand, l992). Annual chickpea yield losses from Fusarium wilt vary from 10 to 15%
(Jalali and Chand, l992) but can result in total loss of the crop under specific conditions
(Halila, 1996; Haware, 1980) but those reports made no assessment as to how factors in the
pathosystem would influence its efficiency. Cortes et al. (1998) demonstrated that the
effectiveness of sowing time as a management practice for Fusarium wilt of chickpea may be
influenced both by virulence of the pathogen race prevalent in soil and susceptibility of the
chickpea cultivar.
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Among fungal diseases in Pakistan, the Fusarium wilt is the most serious disease and has
significance economic importance. From last four to five years, due to prevalence of drought
conditions in the country, this disease is emerging as a devastating problem to chickpea. It
has high saprophytic ability in the soil. The pathogen can survive for a long period during
which it undergoes through different biological competition and environmental stresses
which may lead to the existence of physiologic race (Bendre and Barhate, 1998).
The present study aim is to study the isolation of Aspergillus flavus and Fusarium solani
from Cicer arietinum to understand the pathogen entry, interaction in host to study potential
growth effect after host interaction.
MATERIALS AND METHODS
Glacial acetic acid, sodium hydroxide, potato dextrose agar, antibiotic, potato, dextrose, lacto
phenol, ethanol, plastic pots, phenol, lactic acid, glycerol, cotton blue and other all chemicals
used in this study are haemocytometer, blotter sheets and distilled water used in this
experiment.
Plant material
Seeds of chickpea with three varieties (A.1, KAK-2 and JG-l1) were obtained from the
GKVK, Banglore, Karnataka.
The seeds were subjected into surface-sterilization using 2% sodium hypochlorite for 2 min
to remove the surface contaminants, and then it is rinsed in sterile distilled water and
germinated on filter paper moistened with sterile distilled water which is placed inside sterile
petridishes. 10-l5 seeds are placed inside each plate and incubate 3-4 days at 28 o C and filter
papers are moistened periodically. After one week, germinated seedlings of “A-1”, "KAK-2"
and "JG-l1" were examined under stereomicroscope. Healthy seeds (6 each) were transferred
into 6 earthen pots filled with a red soil. Experiment was done in 3 replicas along with
control. Infected germinated seeds were subjected to detect the presence of seed borne fungi
and symptoms developed on young seedlings was observed.
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Fungal isolate
The two different fungal cultures Aspergillus flavus and Fusarium solani isolated from
chickpeas were further subjected into repeated sub-culture on PDA and incubated for 3-4
days at 28 o C to get sporulated purified culture.
Preparation of fungal inoculum
Potato dextrose broth was inoculated with a conidial suspension of 2 fungal species made to a
final concentration of 10 6 conidia/ml with the aid of Haemocytometer. The suspension was
incubated on a shaker for 34 days at 150-170 rpm and 28 o C. Then culture was filtered
through three autoclaved muslin cloth into a 500-m1 centrifuge bottle. The conidial
suspension centrifuged at 10,000 rpm for 20-30 min at 48 o C. They were washed twice by
resuspending the spores in sterile distilled water and finally resuspended in 2-3 ml of sterile
distilled water. From this step 4-5 ml of conidial suspension at l-2x10 9
conidia/ml was
obtained. The inoculum was prepared fresh at the time of use for all the inoculation
procedures (Lichtenzveig et al. 2006).
Spray inoculation method for anatomy
Spore suspension was freshly prepared according to the method of Lichtenzveig et al. (2006).
The spore concentration was adjusted and ten-day-old seedlings were sprayed with the spore
suspension. Then leaves were collected at various time intervals to understand stages of
disease introgression.
Histology
The leaves were collected at various time intervals (2, 4, 6, 24, 48,72h, 1wk, 2wk, and 10
days after inoculation) then fixed in acetic alcohol (l:3 v/v) and processed further for
histological observations. The fixed material is partially crushed with 3% (w/v) NaOH for 30
mins at 60 o C and thoroughly washed in water for 30 mins to remove NaOH. The washed
leaves were transferred to 5% (wlv) warm cotton blue and stained for 20 min. Finally, the
slides were observed under the microscope and the images were captured with a digital
camera attached to the microscope.
RESULTS
Seed borne pathogens
8 seeds among which seeds were blotted per plate it was found that, all the 8 seeds of JG 11
variety was infected with A. flavus. In A1, 5 among the 8 were found to be infected with the
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same fungus. In KAK- 2, I among the 8 seeds was found to be infected with the fungus. The
results are the mean of three replicas. Thus the common seed borne fungus on all three
varieties was found to be A. flavus. Among the three varieties, JG was more prone to seed
borne infection by A. flavus followed by A1 and KAK2' This A. flavus was cultured on a
PDA and after a week the fungal colony was used to prepare the spore suspension and it was
used to spray the three varieties to study the extent of infection.
Morphological comparison of three different varieties of chickpea
Two different fungal pathogens were isolated from chickpea and subcultured, purified and
morphologically characterized by pathologist, pure cultured and stored for further studies.
Two Aspergillus flavus and Fusarium solani pathogens were inoculated with the chosen three
varieties of chickpea and developed the following symptoms.
JG-11 variety leaves infected with A. flavus turned yellow and the infected area turns brown
with the mycelial colony. There was no prominent spot but the leaf dries up from the sides.
The younger leaves were more prone to infection. Browning was from the tip and also from
the sides. But JG-l1 leaves infected with F. solani shows brownish spots uniform throughout
the leaves. A-1 variety leaves infected with A. flavus showed both dark spots and browning at
the sides even both the leaf surface has got infected. The spots were uniform. But A-1 leaves
infected with F. solani showed very dark spots which were uniform. No infection was
observed on the lower side of the leaves. KAK-2 variety leaves infected with A. flavus
showed wilting, without spots, but only browning at the sides and at the tips, later the leaf
dried up and fall off. The browning areas showed severe mycelial growth. But KAK-2 leaves
infected with F. solani showed a characteristic single spots on and around the mid vein with
less rate of infection. Leaves turned slightly yellow but wilting was not observed. The leaves
remain fresh with the black spot as the infection progress the spots gets a hole in the middle.
Among the three varieties of chickpea which was chosen for study, the A-1 was found to be
more susceptible than the other two varieties. It showed severe wilting and disease symptoms
followed by KAK-2. The JG.11 was found to be resistant compared to others, in case of
KAK-2 and A-1 varieties infected with A. flavus, the infected leaves becomes papery and
falls off with the sporulating fungi which absorbs all the nutrition from the fresh leaves and
utilizes it for its mycelia growth. It makes the cells flaccid at the spot of infection. The plants
infected with A. flavus showed complete wilting within 10 days after infection. The infection
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by the F. solani was not as severe as that of A. flavus. The infection time was delayed
compared to Aspergillus sp,.
Structures progression during infection
The plants were sprayed with the spore suspension and during incubation time, the pathogen
penetration into the host was studied. The entry of the A. flavus spore was found to be
intercellular i.e., in the intercellular spaces via, through the stomata. No special structure like
appresorium was produced by the spore. Thus once attached to the surface the spore
germinates depending on their favourable conditions and it germinated by growing a germ
tube and eventually locates a stoma by a touch responsive process known as Thigmotropism.
The growth ended at the stoma. Then a peg growed into the plant's mesophyll cells. The peg
produced specialised hyphal tips' known as Haustorium which spread around the plant cells
without invading the membranes in order to draws nourishment from the cell which is used
for the hyphal growth of the fungus. The hyphae were thick, long, showed branching, septate,
hyaline and had a sharp tip. The sporulation was observed within a week with many spores.
F. solani spores was also found infecting the three varieties of chickpea and the time of entry
was late and showed less response compared to that of Aspergillus and the hyphal growth
was very slow, hyphae was thin but was very long. The spore upon germination produced
thin hyphae which grew upon the stomata and sends in a branch and the same hyphae
produced another branch laterally so that it also penetrates another stomata, so several
branches are sent in by a single spore which penetrated multiple stomata finally resulting in a
lesion in that particular areas which is morphologically seen as brown spots on the leaves.
Only the mycelial growth was seen after the 2 nd
week of inoculation and it did not sporulate.
Thus, A. flavus was found to be more infectious than the Fusarium in causing infection to the
three chickpea varieties chosen for the study.
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DISCUSSION
Histopathology of the host pathogen interaction is useful in determining the sequence
location and timings of resistance mechanism. F. solani (Mart.) Sacc. Causes Black root
rot/wilt in chickpea. It is one of the most serious disease which causes severe yield loss i.e.,
60-70 per cent under favorable conditions (Tewari and Mukhopadhyay, 2003). It exhibits two
types of symptom such as yellowing and wilting. Initially diseased Plant gets stunted; leaves
turn yellow and are ultimately killed. Haware et al. (1978) reported that internal presence of
F. oxysporum sp. Ciceri through histopathological studies in which Chlamydospore like
structures were observed in the hilum region of seed collected from diseased plants.
Dale and Irwin, (1991) studied the Phytophthora infection on chickpea and found that the
penetration was intercellular occurring between the anticlinal walls of the two epidermal
cells. In this kind of penetration the germ tube was swelled and produced the penetration peg.
The hypha developed inter and intracellularly within the cortex of the root, ramified hyphae
appeared to swell before penetrating cell walls, constricting as they passed through the wall
and regaining their normal diameter after they emerged but here Initial events are adhesion to
the cuticle and directed growth of the germ tube on the plant surface there was no
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appresorium formation instead the spore gets attached to the target cell and started to
germinate into a thick hyphae ending in a sharp Haustorium. In the infection induced by
Fusarium the lateral branches itself act as a haustorium which penetrated the stomata to draw
nourishment from the ce1ls and helped in the hyphal elongation.
The infected plant stem was healthy except the growing tips which had a severe mycelial
growth. As the disease was progressed, the stem and the leaves dried up finally resulted in
wilting. The roots turn black and flaccid'. The root system was rotten and most of the finer
roots destroyed, while the tap roots remained intact, but become dark and necrotic. The plant
finally become brown, dry and wilt. Riahi et al. (1990) studied a quantitative scale for
assessing chickpea reaction to Ascochyta rabiei. Hybrid generations obtained from crosses
between several chickpea cultivars were used to develop a quantitative scale to assess the
reaction of chickpea (Cicer arietinum L.) to A. rabiei under controlled conditions. The
number of lesions, their average and total lengths, and the linear infection index were used to
establish a quantitative scale. The distribution frequency of the number of lesions, average
and total lengths of lesions, and the linear infection index showed that the latter best
described the reaction of chickpea seedlings to A. rabiei. This index provided a satisfactory
separation between resistant and susceptible plants.
The process of seed infection is influenced by the conditions under which the crop grows, the
facts that influenced in the process of infection are: the host and its genotype, the pathogen
and its genotype and environmental facts. An infected seed will not always an infected seed
will be the cause of the infection in the plant which originates, so, it is to say that the
infection caused from an infected seed is an exception, not something to happen generally.
Most of the seed-borne fungus reach and infect the embryo through the flower or from the
peduncle of the fruit, via funiculus. Spores of some fungus reached the stigma and
germinated, producing hyphae that reaches the ovary through the style, where they can stay
as a dormant mycelium until seed germinated and some are capable of infecting the placenta
and penetrate in to the developing ovule or seeds that are still in its formation process and
have not lignified its cover. Penetration through wounds and natural openings can also be
occured (Nome et al, 1996) but in this study it was found that neither such fungal spore nor
the mycelial growth was seen inside the seeds. Fungal spores were seen only on the seed coat
which would have come during the harvest or Seeds packing process because the seeds were
found to be healthy without any infection. As reported by Sitara and Akhter (2007) the
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growth of A. flavus was not reduced by Sodium hypochlorite even at 10% so the A. flavus
infection on the 2% sodium hypochlorite treated chickpea must have come only from the
surface of the inoculated seeds and not from the seeds. So when it was inoculated with the
spores to the young seedling it showed characteristic symptoms if it was already infected with
the fungus it wouldn't have allowed the seeds to germinate nor to reach the seedling
CONCLUSION
Cereals and legumes are the most important sources of plant food for man throughout the
world. Among thousands of known legumes, only 14 species of the grain legumes are
cultivated extensively in different parts of the world for human consumption. Chickpea is a
cash crop and plays a significant role in increasing farm income. The hazardous effects of
chemicals used in plant disease management has diverted plant pathologists to find out the
alternative techniques of plant disease control which may cause little or no adverse effect on
environment. Hence a biological treatment was carried out for structural conformation and
diseases progression was well executed in this study using A. flavus and F. solani to the
chickpea host. Well defined diseases progression was observed. Still application of biological
applied specifically to the seed surface could provide an effective alternative to fungicides as
highlighted.
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