ABSTRACT

Allternaria blight, characterized by necrotic chlorotic lesions is one of the most recalcitrant disease of rapeseed mustard against which natural resistance is yet to be located. Modern biotechnological approaches can be helpful in developing de novo resistance only if molecular basis of host-pathogen interaction of this disease is understood. Studies in our laboratory facilitated identification of a chlorotic toxin (destruxin B) and necrotic toxin (not fully characterized). Besides these phytohormone (s) were also suspected to be significant in the establishment of disease. Interestingly a fraction of extract from infected leaves that could induce roots could also suppress the action of chlorotic toxin on leaf foliar bioassay. The observed antagonistic effects of two structurally different entities viz. pathotoxin and phytohormone on mitotic index of calli from Brassica juncea strongly suggested possibility of involvement of signal transduction cascades in which toxin and phytohoromone converge antagonistically on some common point. Studies on effect of toxin and zeatin on growth of calli of Brassica juncea and induction of p53, a apoptotic protein further support this view. Understanding the mechanism of action of pathotoxin on Brassica species will provide a better appreciation of host pathogen interactions and resistance mechanisms.

Introduction

The productivity of Rape seed mustard, an important oil seed crops in India as well as in other parts of the world, is seriously hampered by Alternaria blight. In the absence of natural resistance gene within brassica species, plant breeders have been so far unable to develop appropriate disease resistant lines. Modern biotechnology methods which can locate, transfer and mutate gene (s) at will can help in development of de novo disease resistance provided that the molecular mechanisms related to phytopathogenesis of Alternaria is delineated.

COMPONENTS OF PATHOGENESIS

The disease, is caused by A. brassicae which is known to produce host selective toxins in vitro that could mimic the typical necrotic and chlorotic symptoms of the disease (Bains and Tewari, 1987; Tyagi 1991). The major component that causes chlorosis has been identified as destruxin B. Different strains of A. brassicae can produce one or more related toxins viz. destruxin B, homodestruxin B and desmethyl destruxin B.

In our laboratory we have recognized more than one components involved in the pathogenesis of alternaria blight. The first clue came from the studies on toxin production by A. brassicae in Fries modified medium in the prsense or absence of yeast extract. Foliar bioassay of isolated crude toxin from spent medium of these culture was carried out on B. napus leaves using 10 μl of each preparation separately. The results indicated that yeast extract supplementation (0.3%W/V) in the synthetic modified Frie`s liquid medium enhanced the yield of chlorotic toxin 5.3 folds and decreased the yield of necrotic toxin 2.6 folds (Table 1) i.e. it increased chlorotic / necrotic ratio in foliar bioassay. There was only 2.6 fold increase in the yield of mycelium. It indicated the possibility of differential regulation at least two seperate toxins under different nutritive conditions.

The toxin (s) was purified by using an approach similar to the one used by Canadian workers (Bains and Tiwari, 1987) assuming that the Alternaria toxin (s) in India is also likely to be destruxin B or its derivatives.

Purification of pathotoxin by gel filtration on sephadex G-10, G-15, LH-20 and subsequently by HPLC gave at least two mutually exclusively toxin viz. necrotic and chlorotic. A highly reproducible and efficient protocol was used for the purification of toxin from isolate P1 of Alternaria brassicae Results are given in Table.2. The amino acid analysis on Pico-Tag system and FTIR spectra confirmed the depsipeptide nature of the Chlorotic toxin, and identified it as destruxin B The initial COSEY and NOSEY analysis backed with mass spectrum indicated that the necrotic toxin could be a polyketide.

HOST SPECIFICITY OF TOXIN

Chlorotic toxin showed a very high degree of host selectivity towards the brassica spp, whereas necrotic toxin showed general toxicity of varying degree against all host and non-host plant leaves used for bioassay (Tyagi, 1991) which included maize, pigeon pea, etc.

The host specificity could not be attributed to a single effect as seen in victorin etc. The molecular and biochemical studies on toxin showed that destruxin B inhibits biosyntheses of all the macromolecules, causes leaching of electrolytes and sugars and brings about aberrations in mitochondria and chloro plast ( Agarwal 1992). Thus the chorotic effect is more like a syndrome as it could not be pin pointed biochemical or molcular action at enzymatic leavel.

TOXIN-PHYTOHORMONE INTERACTION

The possibility of involvement of additional factors most likely phytohormones came from the following facts.

(A) Purification activity jump

Remarkable increase in the yield of activity of chtorotic toxin was during observed during purification on Sephadex G-10. Similary, a drastic decrease in necrotic activity was also observed during purification (Table-2). It was very suggestive that both the chlorotic and necrotic activities could be regulated synergistically or antagonistically by some other compounds. Some inhibitory compound present in crude preparation against the chlorotic toxin was removed during the purification. On the other hand, presence of some additional compounds was perhaps also required to establish necrotic activity.

(B) Green islands Formation

The foliar bioasay of crude toxin on leaf often shows a green zone between necrotic and chlorotic zone. This phenomenon has been seen by earlier workers also ( Mandahar and Suri 1987, Tewari 1993 ).

(C) Induction of rooting and suppression of chlorotic symptoms

Agarwal et al. (1994) observed that crude toxin isolated from Brassica napus purified on sephadex G10 purified toxin fails to demonstrate any chlorotic activity. However, subsequent purification of the same on sephadex LH-20 chromatography yielded L II and L III fractions having chlorotic and root inducing activity, respectively. These suggested that chlorotic in earlier fraction (G-10) was suppressed by a hormone like compound.

(D) Toxin- Phytohormone antagonism

The effects of two structurally different entities viz. pathotoxin and phytohormones was tested by their effect on growth of Callus from Brassica juncea. (Table 3; Pandey 1996). The antagonistic effect seen on mitotic index strongly suggested the role of interactive signaling pathways in pathogenesis of Alternaria blight in Brassica species. Within each lineage the control of cell number is determined by a balance between cell proliferation and cell death. Since phytohormones are associated with cell proliferation and pathotoxins with cell death, it is posible that both toxin and phytohormone converge on cell signal transduction pathway albeit in an antagonistic way.

POSSIBLE ROLE OF CELL SIGNAL TRANSDUCTION PATHWAY

The cell proliferation is a highly regulated process with numerous checks and balances. A ser- Thr kinase (p34 cdc2 ) and its association with different cyclins is deemed to be integral to the cell cycle regulation. Phosphatases and kinases have been also implicated in disease resistance (Felix et al. 1991; Diatrich et al. 1990 ; Felix et al. 1994). Regulation of cell cycle besides being linked to differentiation is also linked to apoptosis through p53. If toxin and phytohormone act through cell signal transduction pathway , as envisaged above, their same effect on the level and / or polymorphism of key components like cdc and p53 ought to be seen.

Studies in our labs have succeeded in detecting differential polymosphic expression of cdc 2 (PSTAIR) in calluses grown under different combinations of phytohormones. Further the levels of p53 were higher in aged callus and senescent leaves so also its level increased dramatically in toxin treated young Brassica juncea leaves or calluses. In the light of the observed antagonism seen between the alternaria toxin and cytokinin (s), these days our group is busy in indentifing those components of cell cycle regulatory machinary which are affected by both of these compounds. This knowledge will help in locating the appropriate factor and develop de novo resistance in Brassica against Alternaria blight.

REFERENCES :

1. Agarwal A. 1992. Ph. D. Thesis submitted to G.B. Pant Univ. of Ag. & Tech. Pantnagar.

2. Agarwal , A. ; Garg G.K. ; Singh U.S. & Mishra, D.P. 1994. Current Science. 66 (6) : 442-443.

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4. Dietrich, A.; Mayers, J.E. and Hahlbrok, K. (1990). J. Biol. Chem. 265, 6360-6368.

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8. Pandey, Dinesh. 1996. MSc. Thesis submitted to G.B. Pant Univ. of Ag. & Tech. Pantnagar. 87 p.

9. Tewari, J.P. 1993. Biochemical basis of resistance to Alternaria brassicae in crucifers. In : Lodha , M.L. ; Mehta , S.L. ; Ramgopal, S ; Srivastava G.P. eds. Adnenees in plant Biotechnology and Biochemistry. New Delhi, Indian Society of Agricultural Biochemists.

10. Tyagi, A.K. 1991. Ph. D. Thesis submitted to G.B. Pant Univ. of Ag. & Tech. Pantnagar

Table 1 : Effect of Y.E. Supplementation on different toxin activity of crude toxin

10 μl of chlorotic toxin preparation was used in the foliar bioassay

Table 2 : Purification of toxin (s) from isolate P₁ of Alternaria brassicae

* One unit of toxin is taken as the amount of toxin in 10 μl preparation that produces a lesion of 1.0 cm2 on B. napus leaf during bioassay.

Table 3 : Effect of Alternaria toxin and Zeatin on mitotic index (GDT).

Each Value is mean of GDT’s of 3 replicates.

Mean values in a colum or row , with same superscript do not differ significantly at cd 5% level.