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CHAPTER 6
PARTIAL CHARACTERIZATION AND BIOASSAY OF CRUDE PHYTOTOXIN
EXTRACT FROM CULTURE FILTRATE OF PHOMOPSIS AZADIRACHTAE
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PARTIAL CHARACTERIZATION AND BIOASSAY OF CRUDE PHYTOTOXIN EXTRACT FROM CULTURE FILTRATE OF
PHOMOPSIS AZADIRACHTAE
INTRODUCTION
Phytotoxins are secondary metabolites produced by plant pathogenic
microorganisms (fungi and bacteria) and are low molecular weight substances. They are
toxic to plants and play an important role in host-pathogen interactions and in disease
expression (Amusa, 2006; Svabova and Lebeda, 2005). During the last decade there is a
remarkable development in the studies on the role of fungal toxins in plant pathogenesis.
Many fungal metabolites are known to be phytotoxic (Desjardins and Hohn, 1997; Yoder,
1980). There is a significant progress in the knowledge of nature, structure and mode of
action of many phytotoxins (Graniti, 1991).
The symptoms induced by the phytotoxic metabolites produced by pathogenic
fungi on their host plants include necrosis, chlorosis, wilting, water soaking and
eventually the death of plants. Pathogens utilize phytotoxins as one of the weapons to
induce disease condition on susceptible host plants (Amusa, 2006). Toxins can act as
suppressors of induced resistance (Graniti, 1991). Pathogenicity or virulence of a
phytopathogen is often attributed to their toxigenicity (Scheffer, 1983). Phytotoxic
metabolites of most of the pathogens play a significant role in pathogenesis (Amusa et
al., 1993; Graniti, 1991).
To regard a metabolite of pathogen as a phytotoxin it should produce an obvious
damage to plant tissue (Amusa, 2006; Scheffer, 1983) when applied at a low
concentration. Phytotoxins are harmful to plants in very low concentrations (Graniti,
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1991). Phytotoxins inhibit the physiological processes in cells surrounding the point of
infection and thereby enabling the spread of disease (Feys and Parker, 2000; Staskawicz
et al., 2001). Phytotoxins act directly on protoplast of the cells. Pathogens that produce
phytotoxins are not affected by the same toxins (Amusa, 2006), while they can result in
electrolyte leakage from the host cells and other modes of toxicity to host plants (Mackay
et al., 1994; Strobel, 1982) and thus helping in disease manifestation. Toxic fungal
metabolites also induce adverse effects on plants such as suppression of seed
germination, malformation and retardation of seedling growth (Lynch and Clark, 1984;
Neergard, 1979).
Investigations on fungal phytotoxins have increased our knowledge about many
facts in plant and fungal physiology, biochemistry, genetics and molecular biology.
Phytotoxins provide an important field of study and research to make substantial progress
towards defining, signaling pathways in defense responses, evolution of pathogen races,
virulence and avirulence factors, the role of programmed cell death in plant disease,
phenomena that distinguish resistant and susceptible phenotypes, evidence for horizontal
gene transfer, disease management strategies and many more (Dunkle, 2005). In cases
where toxins are involved in disease development, the knowledge of such phytotoxins
can be exploited for the control of disease (Amusa, 2006). Now-a-days biocontrol agents
are also screened for their ability to inactivate phytotoxins in addition to control activity.
Pseudomonas spp. and Trichoderma harzianum were reported to detoxify the
anthroquinone, a phytotoxic metabolite, produced by the red rot pathogen Colletotrichum
falcatum Went. (Malathi et al., 2002). Phytotoxic metabolites have been employed in
screening crops for disease resistance (Amusa, 2000; Borras et al., 2001; Crino, 1997;
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Svabova and Lebeda, 2005). With respect to all these matters, knowledge of phytotoxin
chemistry and its role in pathogenesis is important.
Toxigenic pathogen species are present in all main taxonomic groups of fungi
(Svabova and Lebeda, 2005). Production of a wide variety of phytotoxins by many
phytopathogenic fungi was reported (Desjardins and Hohn, 1997; Geraldo et al., 2006;
Jin et al., 1996; Sugawara et al., 1998; Sutherland and Pegg, 1995; Venkatasubbaiah and
Chilton, 1992; Wang et al., 2006; Yoshida et al., 2000; Yu et al., 1990). Phytopathogenic
fungi produce two types of toxins, host-specific and non-host specific toxins (Yoder,
1980). Nedelnik and Repkova (1998) grouped the toxic substances based on several
properties: (1) Chemical characteristics (peptide, terpenoid, glycoside, phenol,
polysaccharide, etc.), (2) Type of the producing organism (fungus or bacterium), (3)
Biological activity (enzyme inhibitor, anti-metabolite, cell-wall degrading substances,
etc.), (4) Host specificity or non specificity.
Many Phomopsis spp. were reported to produce phytotoxins (Avantaggiato et al.,
1999; Horn et al., 1996; Kunwar et al., 1987; Lanigan et al., 1979; Maimone Mancarello
et al., 2005; Mazars et al., 1991; Shankar et al., 1999; Tsantrizos et al., 1992). Culture
filtrates of phytopathogenic fungi are known to contain phytotoxic metabolites (Bashan
and Levy, 1992; Haegi et al., 1994; Jin et al., 1996; Lanigan et al., 1979). Isolation of
toxin from culture filtrates of phytopathogenic fungi was reported (Ahmed et al., 2006;
Bashan et al., 1995; Haegi and Porta-Puglia, 1995; Venkatasubbaiah et al., 1992; Wang,
1986; Zhang and Watson, 2000). In vitro studies on the effect of phytotoxin against host
tissues are carried out using tissue culture (Dahleen and McCormick, 2001; Fernandez et
al., 2000; Gentile et al., 1992; Hollmann et al., 2002; Jaisankar et al., 1999). Screening of
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toxicity on seed germination is also applied as bioassay for phytotoxin and to select
resistant varieties (Amusa, 2006; Gupta et al., 1986; Kunwar et al., 1987; Pakdaman et
al., 2006).
Presence of toxic metabolites in the culture filtrate of P. azadirachtae was
reported by Fathima (2004) and Sateesh (1998). The present investigations were
undertaken to isolate and partially characterize the toxic metabolite from the culture
filtrate of P. azadirachtae and to study the toxicity of that toxin on neem seed
germination and neem callus growth.
MATERIALS AND METHODS
Isolation and culture of the organisms
The Mysore isolate was considered for this study. Isolation of the pathogen from
die-back affected neem twigs was carried out as mentioned in chapter three. Sub-
culturing was done using hyphal tips. Mycelial plugs were removed from margin and
transferred on to fresh potato dextrose agar (Himedia, Mumbai, India) plates amended
with chloramphenicol at 100 ppm. The inoculated plates were incubated for seven days at
26 ± 2oC with 12 h photoperiod. 100 ml of potato dextrose broth (Himedia, Mumbai,
India) was taken in 250 ml Erlenmeyer flask and inoculated with a mycelial agar disc
drawn from advancing margin of seven-day-old cultures. Totally 2.5 l of medium was
inoculated. All the flasks were incubated at 26 ± 2oC with 12 h photoperiod for 25 days.
After incubation, the culture filtrate was filtered through three layers of cheese cloth and
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Whatman No.1 filter paper. Culture filtrate thus collected was filter-sterilized using 0.45
µm membrane filter discs (Sartorius, Goettingen, Germany).
Extraction of toxin
The culture filtrate was concentrated to 10% of its original volume by using a
flash evaporator at 50oC (Stierle et al., 1992; Zhang and Watson, 2000). The concentrated
solution was extracted with equal volume of methanol and then with equal volume of
chloroform so that methanol: chloroform volume is 1: 2 (Filtenborg et al., 1983) with
respect to original culture filtrate volume. The extraction with chloroform was repeated
thrice. The chloroform layer was retained, pooled and evaporated at RT to obtain 914 mg
of dark brownish semi-solid crude extract. The crude extract was dissolved in 9.14 ml of
methanol to have a 10% toxin solution. 8.0 ml of this solution was diluted to 160 ml by
adding the solution drop-wise to double distilled water with continuous stirring to obtain
a stock toxin solution of 5000 ppm having 5% of final methanol concentration (Mackay
et al., 1994). Distilled water with 5% methanol served as stock control solution. These
stock solutions were used for further bioassays. The remaining one ml of crude extract
solution was used for Thin Layer Chromatography (TLC).
Partial characterization of toxin
A) Thin Layer Chromatography (TLC)
TLC was employed using microscopic slides and 20 X 10 cm glass plates with gel
silica (Qualigens, Mumbai, India), without fluorescence indicators. 10 µl of crude extract
was applied in duplicates on slides and developed using different combinations of
chloroform: methanol solvent system (5.0: 5.0 ; 5.5: 4.5 ; 6.0: 4.0 ; 6.5: 3.5 ; 7.0: 3.0 ;
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7.5: 2.5 ; 8.0; 2.0 ; 8.5: 1.5 ; 9.0: 1.0). Then each slide was analyzed under UV light at
365 and 254 nm. Later the plates were exposed under iodine chamber (Sadasivam and
Manickam, 2004). The Rf was calculated using the formula
Distance (cm) moved by the solute from the origin Rf = --------------------------------------------------------------------------------------------------------------
Distance (cm) moved by the solvent from the origin
B) Chemical nature of toxin
The following tests were conducted to know the chemistry of the toxin solution as
per Harborne (1998) and Sadasivam and Manickam (2004). All the chemicals were
procured from Messrs S.D. Fine Chemicals Ltd., Mumbai, India. 20.0 ml of 5000 ppm
toxin solution was diluted to 100.0 ml using sterile distilled water to have 1000 ppm
solution which was used for chemical tests.
Preparation of reagents
1) Mayer’s reagent (Mercuric potassium iodide)
1.358 g of mercuric chloride was dissolved in 60 ml of distilled water. 5.0 g of
potassium iodide was dissolved in 10 ml of distilled water. Both the solutions were mixed
and made up to 100 ml using distilled water.
2) Dragendorff’s reagent (Potassium bismuth iodide)
Solution 1: 0.85 g of basic bismuth nitrate was dissolved in 10 ml acetic acid and
40 ml distilled water.
Solution 2: 8.0 g of potassium iodide was dissolved in 20 ml distilled water.
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5 ml of solution 1, 5 ml of solution 2 were mixed with 20 ml of acetic acid and 100 ml of
distilled water before use.
3) Neutral ferric chloride reagent
1.0 g ferric chloride was dissolved in 100 ml distilled water and neutralized with
sodium hydroxide until a slight precipitate of FeO (OH) was formed.
4) Millon’s reagent was obtained from Qualigens, Mumbai, India.
5) 0.1% Copper sulphate reagent (CuSO4)
0.1 g of copper sulphate was dissolved in 100 ml of distilled water.
6) 10% Sodium hydroxide solution (NaOH)
10 g of sodium hydroxide was dissolved in 100 ml of distilled water.
7) 0.1% Ninhydrin (Triketohydrindene hydrate, C9H4O3.H2O)
0.1 g of ninhydrin powder was dissolved in 100 ml of distilled water.
(i) Tests for alkaloids
a) Mayer’s test
The test solution was treated with Mayer’s reagent and observed for the formation
of a cream colour precipitate which indicates the presence of alkaloids.
b) Dragendorff’s test
The test solution was treated with Dragendorff’s reagent and observed for the
formation of a brown precipitate which indicates the presence of alkaloids.
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(ii) Test for phenolics
Ferric chloride test
To the test solution a few drops of neutral ferric chloride solution was added and
observed for the development of a bluish black colouration that indicates the presence of
phenolic compounds.
(iii) Tests for proteins and amino acids
a) Millon’s test
Two ml of the test solution taken in a test tube was mixed with 2 ml of Millon’s
reagent and boiled gradually over a small flame. The tube was observed for the formation
of a white precipitate which gradually turns red upon heating revealing the presence of
proteins / amino acids.
b) Biuret test
To 2 ml of test solution taken in a test tube, 2 ml of 10% NaOH was added and
mixed well with two drops of 0.1% CuSO4. The tube was observed for the development
of violet or pink colour which is an indication for the presence of proteins.
c) Ninhydrin test
To 4 ml of test solution taken in a test tube, 1ml of 0.1% freshly prepared
ninhydrin solution was added and the tubes were observed for the formation of violet or
purple colour which is an indication for the presence of amino acids.
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Toxicity of crude extract of P. azadirachtae on neem seed germination
20 ml and 10 ml of 5% methanolic stock toxin solution (5000 ppm) were diluted
to 100 ml with double distilled water in separate beakers to obtain 1000 ppm and
500 ppm solutions of toxic metabolites. The toxin solution was filter-sterilized using
0.45 µm filter discs (Sartorius, Goettingen, Germany). Freshly harvested healthy neem
seeds were thoroughly washed, hard endocarp was dissected out and surface-sterilized
using sodium hypochlorite solution (with 5% available chlorine) for 15 min. Then the
seeds were rinsed well in sterile distilled water for five times. About 100 surface-
sterilized neem seeds were treated with 1000 ppm toxin solution for 24 h by placing them
in 25 ml of toxin solution taken in 100 ml beaker. Similarly, the surface-sterilized neem
seeds were treated with 500 ppm toxin solution and control solution separately. Seeds
treated only with the control solution served as control. After treatment the seeds were
germinated by blotter paper and paper towel methods (ISTA, 1993), incubating for
15 days at RT with natural alternate day and night photoperiod. Each treatment had four
replications. Then root length, shoot length and percentage germination were recorded
and the vigour index was calculated using the formula given by Abdul-Baki and
Anderson (1973).
Toxicity of crude extract of P. azadirachtae on neem callus growth
Neem callus cultures were established as per Sateesh (1998). Freshly harvested
seeds were washed in running tap water for 30 min after removing external hard seed
coat. They were surface sterilized with 0.1% aqueous mercuric chloride solution for
15 min and rinsed well in sterile distilled water for five times. The seeds were then
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allowed to germinate on basal Murashige and Skoog (MS) medium (1962) for 15 days.
Then the cotyledonary explants were excised aseptically and transferred to 250 ml tissue
culture bottles having 30 ml of MS medium incorporated with one ppm each of
6-benzylaminopurine (BAP) and Kinetin. These bottles were incubated in 12 h
photoperiod for 30 days. The calli obtained were subcultured and maintained on MS
medium supplemented with same concentrations of hormones and in same incubation
conditions, for every 30 days of incubation. These calli were used for testing the
phytotoxicity of crude extract of P. azadirachtae.
Stock toxin solution was filter-sterilized using 0.45 µm filter discs (Sartorius,
Goettingen, Germany). MS medium having one ppm each of BAP and Kinetin was
amended with different concentrations of crude toxin extract viz., 10, 100, 250, 500 and
1000 ppm separately. Final toxin concentrations were achieved by adding appropriate
volume of 5% methanolic stock toxin solution to a one and half strength sterilized MS
medium (Mackay et al., 1994). About 30 ml of toxin amended MS medium was
transferred to 250 ml tissue culture bottles. Calli from actively growing stage ca. 100 ±
10 mg were transferred aseptically to each bottle. Inoculated bottles having MS medium
amended with control stock solution (1000 ppm) served as control. The inoculated bottles
were incubated as mentioned above. Calli were weighed after 30 days of incubation.
Each treatment had eight replications. The relative growth was calculated (Gowda and
Bhat, 1988) using the formula mentioned below
Final weight – Initial weight
Relative Growth = ------------------------------------------ Initial weight
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RESULTS
Partial characterization of toxin
a) Thin Layer Chromatography
The slides developed with solvent system, chloroform: methanol (7.5 : 2.5)
produced better separation of toxin solution wherein three spots / bands were observed on
the slides and plates . On exposure to UV light at 365 nm the same bands got fluoresced,
in addition to one more band. There was darkening of bands when exposed to iodine
chamber revealing the presence of unsaturated compounds (Fig. 20). The Rf of the bands
are mentioned in table 14.
Table 14. Rf values of different molecules of toxin solution
Sl. No. Rf values
1 0.926
2 0.778
3 0.309
4 0.074
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b) Chemical nature of toxin
(i) Tests for alkaloids
In both Mayer’s test and Dragendorff’s test there was no formation of the
characteristic cream colour and reddish brown precipitate respectively indicating the
absence of alkaloids.
(ii) Test for phenolics
In Ferric chloride test development of a bluish black colouration was not
observed indicating the absence of phenolic compounds.
(iii) Tests for proteins and amino acids
a) Millon’s test
The formation of a white precipitate which gradually turns red upon heating
revealed the presence of proteins / amino acids.
b) Biuret test
The development of violet or pink colour indicated the presence of proteins.
c) Ninhydrin test
The formation of violet or purple colour indicated the presence of amino acids.
In total, the chemical tests indicated the absence of alkaloids and phenolics, and
the presence of amino acids and proteins in the crude toxin extract solution of
P. azadirachtae.
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Toxicity of crude extract of P. azadirachtae on neem seed germination
The germination of neem seeds exposed to both 500 ppm and 1000 ppm
concentrations of toxin solution was completely inhibited wherein the seeds exposed only
to control solution exhibited normal germination (Fig. 21). The root length, shoot length,
per cent germination and vigour index in seeds exposed to control solution are recorded
in table 15.
Table 15. Effect of crude toxin extract of Phomopsis azadirachtae on the germination
of neem seeds
Concentra-tions of Toxin
Root Length (cms)
Shoot Length (cms)
Percentage Germination
Vigour Index
0 10.45 ± 0.10 3.72 ± 0.06 89.50 ± 0.92 1262.42 ± 14.70
500 ppm 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
1000 ppm 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Values are means of four replications ± S.E.
Toxicity of crude extract of P. azadirachtae on neem callus growth
Cotyledonary explants on one ppm of both BAP and kinetin amended MS
medium exhibited good callusing (Fig. 22). Neem calli displayed decreased growth with
increase in the concentration of toxin solution showing a negative proportional relation
(Fig. 24). At 10 ppm calli exhibited the growth almost similar to the control. At 100 ppm
there was decreased growth of calli and at 250 ppm and above in addition to decrease in
the growth, calli also showed browning and necrosis (Fig. 23). Thus with increase in
concentrations of crude toxin extract of P. azadirachtae, pronounced phytotoxic effect on
neem callus was observed.
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116
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DISCUSSION
Plant diseases are the result of the interaction between the host plant, the pathogen
and the environment, which constitute the disease triangle. Phytotoxins play an important
role in the host plant and pathogen interaction. Many deuteromycetous fungi were
reported to release toxic secondary metabolites into the media (Agrios, 2004; Maude,
1996). In the present investigations P. azadirachtae was found to release toxic metabolite
into the medium that was isolated from culture filtrate. The culture filtrates of many
Phomopsis spp. are known to contain toxic metabolites that were purified and
characterized. Horn et al. (1996) isolated phomodiol and phomopsolide B by Phomopsis
spp. Phomopsis helianthi produces cis and trans-4-6-dihydroxymellein and these toxins
contribute to the severity of the sunflower disease caused by P. helianthi (Avantaggiato et
al., 1999). Phomopsis helianthi produces phytotoxin, phomozin (Mazars et al., 1991 and
1990). Maimone Mancarello et al. (2005) reported the production of two phytotoxins of
polyketidic nature by Diaporthe helianthi (anamorph = Phomopsis helianthi). These two
toxins were isolated from culture filtrate of the pathogen and one of the toxins was
identified as phomozin. Phomopsis convolvulus produces three phytotoxins viz.,
convolvulanic acid A and B, convolvulol and convolvulopyrone (Tsantrizos et al., 1992).
Sateesh (1998) reported the production of a toxic metabolite into the medium by
P. azadirachtae, which reduced the seed vigour and seed quality of neem. Culture
filtrates of P. azadirachtae isolates collected from different regions of Karnataka, South
India exhibited different degrees of phytotoxicity against neem seeds by decreasing the
seed vigour and seed quality (Fathima, 2004). Similar effect was observed with culture
filtrates of P. azadirachtae isolates collected from different regions of Tamilnadu, South
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India (Chapter 3). These results reveal the presence of phytotoxic components in the
culture filtrate of P. azadirachtae. Lanigan et al. (1979) were able to isolate phomopsin A
only from culture filtrate.
Methanol and chloroform solvents used for the extraction of toxic metabolite
from culture filtrate proved to be beneficial and is in agreement with the earlier reports.
Lanigan et al. (1979) and Shankar et al. (1999) reported isolation of Phomopsin A and
other phomopsins using methanol. Methanol: chloroform (1:2) solvent system was most
efficient for mycotoxin extraction from fungi (Filtenborg et al., 1983). Utilization of
methanol and chloroform for mycotoxin extraction was reported (da Motta and Vanlente
Soares, 2000; Sugawara et al., 1998; Vidyasekaran et al., 1997; Zhang and Watson,
2000). Kurt (2004) utilized methanol as elution solvent to purify toxin from culture
filtrate of Corynespora cassicola. Phytotoxins produced by Exserohilum monoceras was
extracted from culture filtrate using chloroform (Zhang and Watson, 2000).
Thin Layer Chromatography method and UV visualization is used to detect,
identify and partially purify mycotoxins (Filtenborg et al., 1983; Geraldo et al., 2006;
Scott et al., 1970; Steyn, 1969; Zhang and Watson, 2000). Analysis of fluorescence
produced by certain compounds is one of the methods available for the evaluation of
crude extracts. Many substances which do not fluoresce in ordinary light emit radiation
when exposed to UV light (200 - 400nm) (Mahadevan, 2001). Owing to this concept the
above phenomenon was employed in the present study to locate the Rf values of the
eluted bands from the extract. Chemical tests have revealed that the crude extract
contains amino acids and protein but no alkaloids and phenolics. Phytotoxins are known
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to be low molecular weight peptides, terpenoids, phenolics, glycosides or carbohydrates
(Nedelnik and Repkova, 1998; Strobel, 1982; Walton and Panaccione, 1993).
The toxicity of P. azadirachtae crude extract was investigated by its effect on
seed germination and quality, wherein the toxin completely inhibited the seed
germination and significantly reduced the quality of seed. This is in accordance with
other similar observations. Filter-sterile culture filtrates of Diaporthe phaseolorum var.
sojae (Phomopsis sojae) inhibited germination of cabbage (Brassica oleracea),
cantaloupe (Cucumis melo), onion (Allium cepa), soybean (Glycine max) and wheat
(Triticum vulgare) seeds within 24 h of incubation (Kunwar et al., 1987). Gupta et al.
(1986) reported the phytotoxicity of culture filtrate of Alternaria porri on seed
germination and seedling vigour of onion. Secalonic acid A (SAA) isolated from
Pyrenochaeta terrestris and Penicillium oxacilum inhibited onion seedling elongation at
very low concentrations (Zeng et al., 2001). Pakdaman et al. (2006) observed inhibition
of germination of wheat seeds on Fusarium graminearum phytotoxin-containing agar
medium.
The phytoxicity of P. azadirachtae crude extract was also evaluated by its effect
on neem callus. Tissue culture technique provides a controlled environment, where the
effect of toxin or any chemical can be evaluated on callus tissues without any interfering
external biotic and abiotic factors (Gowda, 1988). Callus tissues are more sensitive than
intact plants. Thus the tissue culture technique provides a good experimental tool for
precise evaluation of the phytotoxicity of fungal toxic metabolites in vitro (Sateesh,
1998). The good yield of callus tissues of neem with kinetin and BAP at one ppm is par
with that of Sateesh (1998). Various workers reported the establishment of callus tissues
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in neem (Gautham et al., 1993; Kearney et al., 1994; Ramesh and Padhya, 1988; Sateesh,
1998). Exposure of neem callus to different concentrations of P. azadirachtae toxic
metabolite revealed its phytotoxicity against neem tissues. A progressive decrease in the
callus growth observed with the increasing concentration of toxin is in conformity with
the previous reports. Final dry weights of Cucumis melo (muskmelon) callus exposed to
rosidin-A toxin of Myrothecium roridum showed an inverse relationship towards toxin
concentration (Mackay et al., 1994). Decrease in logarithmic growth rates of tobacco
callus tissues by T-2 toxin of Fusarium tricinctum was observed. Toxin concentration of
0.003 µm decreased growth rate while a concentration of 0.081 µm completely inhibited
the growth of callus (Helgeson and Haberlach, 1973). Mohanraj et al. (2003) observed
toxicity of Colletotrichum falcatum phytotoxin on the sugarcane callus. Corn callus
growth was inhibited by Helminthosporium carbonum race 1 toxin (Wolf and Earle,
1990). van Asch et al. (1992) reported the phytotoxicity of fumonisin B1, moniliformin
and T-2 toxin from Fusarium sp. on corn callus tissue.
The callus tissue undergoes necrosis and brownish discoloration because of the
accumulation of phenolic compounds and their products (Mohanraj et al., 2003). Similar
browning of callus tissues was observed in the present studies. Cvikrova et al. (1992)
reported accumulation of phenolic acids in alfalfa cell cultures exposed to culture filtrate
of Fusarium oxysporum. Exposure of Coffea arabica callus to phytotoxic culture filtrates
from Colletotrichum coffeanum resulted in reduced growth and necrosis of callus
(Nyange et al., 1995). Other changes in callus that were reported on exposure to
phytotoxins include changes in permeability, protein pattern, electrolyte leakage,
inhibition of shoot difference and loss of chlorophyll (Gonza Lez et al., 2000; Mackay et
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al., 1994; Mohanraj et al., 2003; Ramos and Maribona, 1991). The reduction in the callus
tissue quality that was observed in the present study with the increase in toxin
concentration may be attributed to some of the phytotoxic effects mentioned above.
Toxins are used for screening of resistance, selection for resistance, in tissue
culture (Amusa, 2006; Behnke, 1980; Dozet and Vasic, 1995; Jaisankar and Litz, 1998;
Vidhyasekaran et al., 1990). Tissue culture techniques have produced germplasm with
enhanced disease resistance (Daub, 1986). Growing the plant callus in the presence of a
fungal culture filtrate or purified fungal toxin is widely used for the selection of disease
resistant lines (Sacristan, 1982). Similarly the phytotoxin of P. azadirachtae could be
used for the selection of die-back resistant lines of neem employing tissue culture
technique.
The results of present studies revealed the ability of P. azadirachtae to produce
phytotoxic compound in the culture filtrate and its toxicity on neem tissues. Thus the
involvement of this toxin in the development of die-back symptoms is a possibility.
Proper understanding of toxin chemistry and its role in pathogenesis requires further
investigations and the current investigations provide a proper base for this.
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