T H E E N Z Y M I C A C T I V I T Y O F M A C R O P H O M I N A P H A S E O L I , (Maubl.), A s h b y

BY (MISS) K. RADHA

(Junior Research Fellow, University Botany Laboratory, Madras-5)

Received July 22, 1953 (Communicated by Prof. T. S. Sadasivan, ~.A.SC.)

THE nature and role of pectic enzymes in fungal diseases of plants were dealt with in detail by Brown (1915) and Davidson and Williaman (1927). The present communication relates to the pectolytic activity of Macrophomina phaseoli with reference to frenchbean plants and also the loss of coherence observed in cut pieces of potato by enzyme extracts in the fungal mat.

EXPERIMENTAL

Pectin Content of Healthy and Diseased Frenchbean Plants

Earlier studies have revealed the consistent maceration of the tissues of frenchbean plants infected by M. phaseoli. Since pectin forms the important constituent of the middle lamella of the plant cell it was considered worth- while to study the degradation of pectin in diseased frenchbean plants (arti- ficially infected with M. phaseoli--a single pycnidiospore culture of the fungus isolated from a naturally infected frenchbean plant). The following procedure was employed for the estimation of the pectin of frenchbean plants : -

The plant material was collected 3 days after wilting and 3 g. samples, containing 7 to 8 plants without leaves and root system, were used for each estimation following the modified method adopted by Nanji and Norman 0928) of the original calcium pectate method of Carre and Haynes. Simul- taneously estimations of the pectin content of the correspondingly healthy plants were carried out.

TABLE I

Percentage pectin content of frenchbean plants, healthy and diseased

Replicates Mean

Diseased 1.36 t 1.09 1.283 ii 1.4

Healthy 2.41 2.44 I 2.54 2.466

B~ 231

232 (MIss) K. RADHA

The results presented in Table I show that the pectin content of the diseased plants is only about 50~o that of the healthy ones, and it was there- fore decided to study the enzyme degradation of pectin in vitro by enzyme extracts of the fungal mat.

The Loss of Coherence in Potato Tissue by M. phaseoli

In the present in vitro studies on the enzymic activity of M. phaseoli the fungus was grown for 48 hours in frenchbean plant extract (Frenchbean plants 200g., d;stilled water 1,000ml. autoclaved at 15 lb. pressure for 15 minutes).

Enzymic Preparations

(1) Endo-cellular Enzyme.--The fungal mat was harvested after 3 days growth, washed in distilled water and filtered over Buchner funnel, pressed between folds of filter-paper and the wet mycelium extracted with phosphate buffer (KH~PO4-NaOH) at the following pH levels--pH 5.8, 6.4, 7-0 and 7.6--20 mg. of the wet fungal mat was thoroughly macerated in 20 ml. of buffer and centrifuged at 3000 revolutions for 20 minutes. Both the centrifugate and the mycelial residue suspended in the same buffer were tested for the endo-cellular enzyme activity by Brown's (1915) method, to assess the completeness of the extraction of the enzyme.

(2) Exo-CelMar Enzyme.--The culture filtrate after the growth of the fungus was dialyzed and tested in the same way for the presence of the exo- cellular enzyme activity.

The time taken by the different preparations to macerate the substrate (potato tissue) at 37 ~ C. are presented in Table II.

The maceration of the potato tissue observed in the enzyme preparations resembling the condition occurring in the plants during pathogenesis is possibly due to the activity of the fungal enzyme since no such effect was produced either by water or buffer solutions or the inactivated enzyme preparation. The lack of the macerating capacity in the mycelial residue of the dry de- hydrated mat (with acetone and ether) indicates that the macerating effect o f the mycelial residue of the wet mat was due to the enzymic activity of the fungus remaining alive in the test suspension and not due to incomplete extraction. The exo-cellular enzyme present in the fungal filtrate had poor activity, probably because it was present in low concentrations, pH 6.4 of the buffer was found to be optimum for extracting the enzyme and further experiments were carried out at that pH.

Enzymic Activity of Macrophomina phaseoli

TABLE II

233

Enzyme preparations

Wet mycelial extracts in buffer solution

Wet mycelial residue in buffer solution

Wet mycelial extract in water

Wet mycelial residue in water

Dialized culture filtrate

Inactivated extract

Dehydrated mycelial extract in buffer solution

Dehydrated mycelial residue in buffer solution

pH

5.8

Time in hours

No maceration after 40 hours

6.4 16

7-0 40

7.6 40

5.8 40

6 .4 4O

7.0 40

40 7.6

Variable pH recorded between 6.8-7

do

do

6.4

20

16

Poor maceration after 40 hours

Nil

16

Nil

Controls

Buffer Water

Nil Nil

Nil Nil

40 Nil

6 .4

40 Nil

Observations of repeated trials show that in the higher concentration of the extract the enzymic activity was more pronounced as lesser time was taken to macerate the substrate. The coherenace of the potato tissue was lost in 16 hours, 7 hours and 3 hours in fungal extracts of 20 rag., 250 mg. and 500 mg. of wet mat in 20 ml. of buffer respectively.

DISCUSSION

Experimental results suggest that M.phaseoli belongs to the group of fungi whose pathogenesis is related to the production of pectic enzymes which attack the middle lamella and result in the loss of coherence of tissue. It is quite probable that the fungal extract may contain enzymes, other than

234 (MIss) K. RADHA

pectic enzymes, playing important role in the pathogenesis. Convincing evidence of the existence of three distinct pectic enzymes namely proto- pectinase, pectase and pectinase was recorded in fungi, viz., Rhizopus tritici, Sclerotium cinerea and Botrytis cinerea (although all three of them were not produced by the same fungus) by Davidson and Williaman (1927). Further work on the enzymic activity of the extracts on pectin and related substances is underway to identify the enzymic constituents of the fungal extracts with these and will be reported later. The experimental results of low pectin content of frenchbean plants infected with M. phaseoli (Table I) resulting in complete loss of coherence suggest that the fungus definitely produces pectolytic enzymes during plant infection.

SUMMARY

1. The pectin content of frenchbean plants infected with M. phaseoli was 50% less than that of correspondingly healthy plants.

2. That the loss of pectin in the diseased frenchbean plants was due to peetolytic enzyme activity of the pathogen was proved by in vitro studies on cut bits of potato with enzymic extracts of the fungus.

ACKNOWLEDGMENTS

The author is greatly indebted to Prof. T. S. Sadasivan for the help and guidance rendered during the course of this investigation. She wishes to thank Dr. C.V. Subramanian and Mr. K. Lakshminarayanan of the Uni- versity Botany Laboratory, Madras, for critical reading of the manuscript. Her thanks are also due to the Government of India for the award of a Research Scholarship and the University of Madras for the facilities afforded to carry out this work.

1. Brown, W.

2. Davidson, F. R. and Wilfiaman, J. J.

3. Nanji, D. R. and Norman, A .G .

REFERENCES

"Studies in the physiology of parasitism, I. The action of Botrytis cinerea," Ann. Bot., 1915, 29, 313--43.

"Biochemistry of plant diseases. IX. Pectic Enzymes," Bot. Gaz., 1927, 83, 329-61.

"Studies on Pectin, II. The estimation of the individual pectic substances in nature," Biochem. J., 1928, 22, 596-604.