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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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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.