Cultural, Morphological and Biochemical Variability among the Isolates of Phomopsis azadirachtae from Karnataka
S. Shankara Bhat
A study on cultural, morphological and biochemical variability among the isolates of Phomopsis azadirachtae Sateesh, Bhat and Devaki collected from sixteen districts of Karnataka state, South India was carried out. The isolates were compared for the cultural and biochemical variability. It was observed that the isolates varied considerably for the cultural characteristics such as mycelial growth pattern, colour of the colony, sporulation behaviour, texture of pycnidia, production of alpha and beta conidia and the dimension of conidia produced. Estimation of water soluble mycelial proteins and toxicity of culture filtrate of P. azadirachtae isolates revealed a remarkable difference in the mycelial protein content of the isolates and different isolates exhibited various extent of toxicity on the germination of neem seeds.
Received: November 01, 2011;
Accepted: March 05, 2012;
Published: June 05, 2012
Neem (Azadirachta indica A. Juss.) a member of mahogany family Meliaceae
is well-known for its biomedical properties. It is an evergreen, ecofriendly,
native tree of Indian sub-continent. Neem is a source of effective bio-pesticides
and provides a cure for many ailments right from common cold to cancer and AIDS
(Anonymous, 2009; Girish and Bhat,
2008a). The eco-friendly neem tree, in spite of having antimicrobial properties,
is infected by various pathogens belonging to bacteria and fungi. The most destructive
pathogen of neem at present is Phomopsis azadirachtae Sateesh, Bhat and
Devaki which causes die-back disease (Sateesh et al.,
1997). The chief symptoms of the disease are twig blight, inflorescence
blight and fruit rot. The disease results in almost 100% loss of fruit production
(Bhat et al., 1998; Girish
and Bhat, 2008b), because of which, neem seeds used as a raw material in
the preparation of bio-pesticides, medicines and various industrial products
are not obtained.
Plant pathogens are important components of the biodiversity of all natural
ecosystems. Remarkable differences in cultural, morphological and biochemical
characteristics are observed among pathogen populations from different geographical
locations (Thakur, 1999). Differences in cultural, morphological
and biochemical characteristics have been reported among populations of various
plant pathogens (Sharma et al., 2002; Basandrai
et al., 2005; Khurana et al., 2005;
Hosen et al., 2010). Phytopathogenic fungi exhibit
intraspecific variability in toxin production (Nandakumar
et al., 2007; Asran and Amal, 2011; Singh
and Kumar, 2011).
A good number of Phomopsis species are reported to exhibit remarkable
intraspecific variability (Brayford, 1990; Shivas
et al., 1991). Die-back disease of neem caused by P. azadirachtae
is widespread in different parts of Karnataka State, South India (Sateesh,
1998). Girish and Bhat (2010) reported the presence
of morphological, cultural and biochemical variability among the P. azadirachtae
collected from different regions of Tamil Nadu state, India. Significant
differences were found in the protein banding patterns of P. azadirachtae
isolates from Karnataka (Fathima et al., 2004)
and Tamil Nadu (Girish et al., 2009), India.
The present investigations were undertaken to study the morphological, cultural
and biochemical variability among the isolates of P. azadirachtae collected
from 16 districts of Karnataka State.
MATERIALS AND METHODS
Sites sampled: The collections of infected neem twig samples were made from Mandya, Gulbarga, Mysore, Bijapur, Hassan, Chikmagalur, Shimoga, Kolar, Raichur, Tumkur, Chamarajnagar, Chitradurga, Belgaum, Davanagere, Bengaluru and Bellary. The twigs were either used immediately after their collection or stored in brown paper bags or polythene covers in a refrigerator at 4°C until used for further study.
Isolation of P. azadirachtae from infected neem shoot: The infected
twigs collected from each district were cut into 2-3 cm pieces with middle transition
region of healthy and infected portion. Healthy twig explants served as control.
Both healthy and infected twig pieces were washed separately with running tap
water for one hour. Later they were cut into 1-1.5 cm segments with transition
zone at the middle portion. The bark was removed and the segments were washed
thoroughly and were surface-sterilized using sodium hypochlorite solution (with
5% available chlorine) (Sauer and Burroughs, 1986). Then
they were rinsed six times with sterile distilled water. The explants were plated
on Potato Dextrose Agar (PDA) medium amended with 200 ppm of chloramphenicol
in Petri plates at the rate of four segments per plate (Sateesh,
1998). The inoculated Petri plates were incubated for 3-7 days in dark and
observed for the growth of the pathogen from the twig segments (Alexopoulos
et al., 1996). Later the plates were kept near the window on the
laboratory bench for natural photoperiod and the pathogen was allowed to sporulate.
Mycelial plugs (5 mm diam.) were removed from the advancing margins of 7 day
old cultures of each isolate and transferred onto fresh PDA in Petri plates
(90 mm diam.). Three replicates of each isolate were maintained and the plates
were incubated for 10 days at 26±2°C for 12 h photoperiod. The isolates
were identified as per Sateesh et al. (1997).
The isolates were designated as follows: Mandya-Pa 01, Gulbarga-Pa 02, Mysore-Pa
03, Bijapur-Pa 04, Hassan-Pa 05, Chikmagalur-Pa 06, Shimoga-Pa 07, Kolar-Pa
08, Raichur-Pa 09, Tumkur-Pa 10, Chamarajnagar-Pa 11, Chitradurga-Pa 12, Belgaum-Pa
13, Davanagere-Pa 14, Bengaluru-Pa 15 and Bellary-Pa 16.
Cultural characteristics: Mycelial plugs were removed from the advancing
margins of 7 day old cultures of each isolate and transferred onto fresh PDA
plates. Three replicates of each isolate were maintained and incubated for 10
days at room temperature (26±2°C) with 12 h photoperiod. The single
spore culture (pure culture) was obtained on PDA medium by inoculating a single
spore (Tuite, 1969). The cultures thus obtained were compared
for their cultural characteristics. The colony morphology was assessed for growth
characteristics, mycelial type, colour of the colony, colony diameter and texture
and growth pattern of pycnidia. To measure the conidial dimension, the spores
were collected onto the microscope slides, by touching the spore ooze. They
were spread by adding a drop of cotton blue in lactophenol. About 100 alpha
(α) and 100 beta (β)-conidia were measured in case of each isolate
(Uecker and Caruso, 1988).
Estimation of water soluble mycelial proteins of P. azadirachtae
isolates: All the 16 isolates of P. azadirachtae were grown separately
on Czapek Dox broth for seven days. Mycelial mat was taken out, washed thoroughly
with distilled water and dried using filter paper. One gram of the dried mycelium
of each isolate was homogenized in 10 mL of TCA and centrifuged at 3000 xg for
20 min. The residue was re-dissolved in 10 mL of phosphate buffer and centrifuged.
The supernatant was used for protein estimation as per Lowry
et al. (1951).
Effects of culture filtrate of P. azadirachtae isolates on germination
of neem seeds: To determine the variability in toxicity of culture filtrate
among the isolates of P. azadirachtae, the filtrate of each isolate was
obtained from 25 day old culture grown on potato dextrose broth. About 100 healthy
surface-sterilized neem seeds were treated with the culture filtrate of each
isolate separately by incubating in a beaker for 24 h. The control treatment
included medium alone and sterile distilled water. The seeds were plated on
sterile moist blotter in petri plates or on standard paper towels (ISTA,
1993) and incubated at room temperature (26±2°C). Each treatment
had four replications. After 10 days shoot length, root length and percentage
germination were recorded. Vigour index was calculated using the formula given
by Abdul-Baki and Anderson (1973).
Sites sampled and isolation of P. azadirachtae from infected neem
shoots: The field survey of various districts of Karnataka state, South
India revealed the occurrence of die-back disease in all the 16 districts of
Karnataka state, South India. The infected neem segments collected from all
the locations developed fungal colonies whose identification was confirmed as
P. azadirachtae as per Sateesh et al. (1997).
All the isolates produced characteristic α and β-conidia.
Cultural characteristics: The isolates of P. azadirachtae derived
from naturally infected neem shoots exhibited variable colony characteristics
on PDA plates at 26±2°C. The isolates varied considerably for mycelial
growth, colour of the colony, sporulation behaviour, growth pattern, texture
and formation of pycnidia, production of alpha and beta conidia and their length
and breadth (Fig. 1). The cultural characteristics of ten
of the isolates are presented in the Table 1. Isolate Pa 14
had the longest alpha conidia (11.2x3.0 μm) while Pa 09 had the shortest
alpha conidia (4.2x1.0 μm). Isolate Pa 04 had the longest beta conidia
(24.2x1.8 μm) while shortest beta conidia (15.3x1.4 μm) were observed
in isolate Pa 13. The isolates also varied significantly for colony growth and
sporulation rate. Isolate Pa 07 had the largest colony (74 mm diam.) while Pa
06 had the smallest (32 mm diam) (Fig. 2).
||Ten day old colonies of P. azadirachtae collected from
different regions of Karnataka on potato dextrose agar
Maximum sporulation was observed in Pa 08 (280 to 300 pycnidia/colony), while
it was minimum in Pa 11 (25 to 40 pycnidia/colony) (Fig. 3).
Further almost all the isolates produced more number of α conidia than
β-conidia except the isolate Pa 04 which produced large number of beta
conidia and very few alpha conidia with an alpha to beta conidia ratio of 8:42
unlike rest of the 15 isolates in which the ratio ranged between 35:15 to 44:6.
Estimation of water soluble mycelial proteins of P. azadirachtae
isolates: The isolates of P. azadirachtae showed significant difference
in their mycelial protein content.
||Colony characteristics of the 10 isolates of P. azadirachtae
on potato dextrose agar plates after 10 days of incubation at 26±2°C
||Colony diameter of 16 isolates of P. azadirachtae on
potato dextrose agar medium after 10 days of incubation at 26°C, Bars
indicate standard deviation
||Sporulation rate of 16 isolates of P. azadirachtae
on potato dextrose agar medium after 10 days of incubation at 26°C,
Bars indicate standard deviation
|| Water soluble mycelial protein of 16 isolates of P. azadirachtae
|Values given are mean of three replicates ±SE, Values
followed by different superscript letters differ significantly when subjected
to Tukeys HSD (honestly significant difference)
The protein content of different P. azadirachtae isolates is presented
in Table 2. Isolate Pa 04 had the highest mycelial protein
content 150 μg g-1 whereas the isolate Pa 11 had the lowest
mycelial protein content 85 μg g-1.
|| Effect of culture filtrate of 16 isolates of P. azadirachtae
on germination of neem seeds
|Values given are means of three replicates ±SE, Values
followed by different superscript letters differ significantly when subjected
to Tukeys HSD (honestly significant difference)
Effects of culture filtrate of P. azadirachtae isolates on germination of neem seeds: The neem seeds treated with culture filtrate of isolates of P. azadirachtae collected from various districts showed reduction in their germination and seed vigour when compared to control seeds in both Petri plates and paper towels. Further it was observed that the culture filtrate of different isolates exhibited different extent of toxicity on the germination of neem seeds. The difference in the level of toxicity is presented in the Table 3. The seeds treated with isolate Pa 03 culture filtrate showed lowest percentage of germination and the seedling quality was also significantly affected. Isolate Pa 09 culture filtrate was least toxic compared to all other isolates.
Fungi are a unique group of microorganisms with their wide range of adaptability
in different ecological situations (Moore, 1996). The
genus Phomopsis is important plant pathogen (Raeisi
et al., 2011) and reported to be a highly variable pathogen (Brayford,
1990; Shivas et al., 1991; Akhtar
and Chaube, 2002). The present study revealed remarkable differences in
cultural, morphological and biochemical characteristics of various P. azadirachtae
isolates collected from different agroclimatic regions of Karnataka State, South
India. Similar differences were observed among the P. azadirachtae isolates
of Tamil Nadu, in radial growth, colony morphology, colour, texture, pycnidial
density and arrangement, conidial number and size (Girish
and Bhat, 2010). Similar variation was reported in Phomopsis oblonga
isolated from Ulmus species in the British Isles and Italy (Brayford,
1990). Intraspecific variation was demonstrated among the isolates of Phomopsis
leptostromiformis the causal agent of stem canker on Lupinus angustifolius
using cultural and biochemical techniques (Shivas et
al., 1991). Similar variability among the isolates of other phytopathogenic
fungi was also reported. Rhynchosporium secalis isolates from barley
in different agroecological zones of Ethiopia differed markedly in several cultural
characteristics like colony and conidial morphology, colony growth rate and
sporulation (Meles et al., 2004). Isolates of
Sclerotinia sclerotiorum collected from infected lentil plants from two
agroecological zones of Syria showed considerable variation in cultural characteristics
such as mycelia growth, mycelia pigmentation and sclerotial production (Akem
et al., 2006). Two isolates of Pythium ultimum var. ultimum
isolated from Egypt and Germany varied in the size of sexual organs, oospore
production, growth rate and other morphological and physiological characteristics
(Al-Sheikh and Abdelzaher, 2010).
The present investigations revealed that in one of the isolates of P. azadirachtae,
designated as Pa 04 collected from Bijapur district, there was abundant production
of beta conidia. Generally beta conidia production is influenced markedly by
the substrate (Parmeter, 1958). The remarkable difference
in alpha to beta conidia ratio in the isolate Pa 04 in the present study could
be possibly due to the difference in the host growing in different geographical
Studies on non-morphological characteristics such as wall composition, proteins
and other hydrocarbons, of fungal isolates becomes important to differentiate
among isolates when the strains no longer produce typical morphological structure
(Jernejc and Cimerman, 2001). A remarkable variation
in the mycelial protein content of various isolates of P. azadirachtae was
observed in the present study. Similar observations were made by Girish
and Bhat (2010) among the Tamil Nadu isolates of P. azadirachtae
and Khurana et al. (2005) among the Alternaria
brassicae isolates. The variations could be possibly due to the difference
in the geographical locations from where the isolates are collected.
A good majority of deuteromycetes are reported to release toxic secondary metabolites
into the media (Maude, 1996; Agrios,
2004). Sateesh, (1998) indicated the release of a
toxic metabolite by P. azadirachtae into the medium which reduces the
seed vigour and seed quality, based on the study of effects of culture filtrate
on germination of neem seeds. In the present investigations, a remarkable difference
in toxicity of culture filtrate was observed among various isolates of P.
azadirachtae on the germination of neem seeds. Similar observation was reported
by Shivas et al. (1991) among Phomopsis leptostromiformis
isolates. Variation in toxin production among the isolates of Sarocladium
oryzae causing rice sheath blight was reported by Nandakumar
et al. (2007). Isolates of Fusarium moniliforme from pepper
plants produced different quantities of each of fumonisin, zearelenone in
vitro (Abo-Elnaga and Ahmed, 2007). A remarkable
difference in toxin production was observed among the isolates of Fusarium
oxysporum f. sp. chrysanthemi pathogenic to Chrysanthemum
(Singh and Kumar, 2011).
The results of present study suggest that P. azadirachtae is a highly
variable pathogen. These variations can be attributed to the influence of host
variety and prevailing environment. This variability in pathogen populations
makes them capable of invading a wide variety of host plants, thereby reducing
the possibility of evolving disease management methods (Moore,
1996). The primary objective of studies on variation in pathogenicity or
virulence is breeding and exploitation of resistance for disease management.
The study of variations among the fungal isolates will also be helpful in characterizing
sub-specific taxa and thereby in fungal taxonomy (Jernejc
and Cimerman, 2001).
The authors thank University of Mysore for facilities. Syeda Kousar Fathima acknowledges UGC, New Delhi for granting FIP fellowship.
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