In vitro and in vivo production of polygalacturonase , polymethylgalacturonase and cellulase enzymes by Alternaria solani at different incubation periods

The production of polygalacturonase (PG), polymethylgalacturonase (PMG) and cellulase (Cx) enzymes have been studied at different incubation period. The fruit rot pathogen of tomato Alternaria solani was cultured at 28°C on semi ripe tomato fruit broth medium for 2, 4, 6, 8, 10 and 12 days. Isolated pathogen A. solani has a capability of producing PG, PMG and Cx enzyme within short incubation period, that is, 2 days incubation. Production of these enzymes gradually increased by increasing the length of incubation period up to 6 days and further increase in length of incubation up to 12 days, did not show any effect on the production of PG, PMG and Cx. The 6 days incubation was found to be the best incubation period for the production of all these three enzymes. The production of PG and Cx was also more in comparison with PMG enzyme. The enzyme activity in healthy and diseased semi ripe tomato fruits was also assayed at different incubation periods and it was found to be maximum in diseased tomato fruits in comparison with healthy tomato fruits. It was also found that PG and PMG enzymes were produced in higher concentration than the Cx enzyme. Among six incubation periods, 6 days incubation period was found best for maximum production of all these three enzymes.


INTRODUCTION
Fungal diseases of fruit and vegetable plants are known to cause great damages all over the world.Tomato (Lycopersicon esculentum Mill.) is the most ancient among the vegetable fruits.Among the fungal diseases, fruit rot is the most severe disease of tomato, which is caused by Alternaria solani (Mehta, 1973;Chaurasia, 2001;Chaurasia andChaurasia, 2010 andChaurasia et.al., 2013a).
Pectin is present in the middle lamella of cell wall, in the form of magnesium and calcium pectate.They are complex and colloidal in nature and mostly comprises of anhydroglacturonic acid units, linked together by α-1,4 glycosidic linkages to form a chain like structure of polygalacturonic acid units.The middle lamella is degraded by pectolytic enzymes secreted by the phytopathogens.However, the production and activity of pectolytic enzymes by fungal pathogens is an important process of pathogenesis (Gupta, 1956;Wood, 1960;Brown, 1965;Bateman and Millar, 1966;Mehrotra et al., 1988).
Next to pectin, the cellulose is the major component and basic unit of structural framework of plant cell walls, which act as barrier.The pathogens feed successfully only after breaking this barrier with the help of cellulolytic enzyme.Cellulolytic enzymes secreted by pathogen play on important role on living plant tissues in softening them and may further participate indirectly by hydrolysing cellulose into soluble saccharides which serve as food for pathogen.The production of cellulolytic enzymes and degradation of cellulose by several fungi has been studied by many workers like Reese (1956Reese ( , 1963)), Basu and Ghose (1960), Gascoigne and Gascoigne (1960), Bateman (1964), Norkrans (1967), Singh and Singh (1988), Kaur et al. (1989) and Sohail et al. (2011).Production of cellulolytic enzymes by several pathogenic fungi and its role in the development of diseases has also been reported (Lucas and Sherwood, 1966;Hasija and Batra, 1982;Sanghi and Rajak, 1987;Singh et al., 1991;Singh and Shukla, 1999;Sharma, 2000;Chaurasia, 2001;Shukla and Dwivedi, 2012;Jat et al., 2013, Chaurasia et al., 2013b, c).
Keeping the above facts in mind, in the present paper, the production and activity of pectolytic and cellulolytic enzymes by A. solani in culture and in the healthy and diseased tomato fruits at different incubation periods have been investigated.

Organism
A. solani (Ellis & Marts) Jones & Grout was isolated from the diseased fruits of tomato (Chaurasia and Chaurasia, 2010).The organism was maintained on potato dextrose agar slants.

Production of enzymes in vitro
The semi ripe tomato fruit medium tested by Chaurasia et al. (2013b) was used to study polygalacturonase (PG), polymethyl galacturonase (PMG) and cellulase (Cx) enzyme production in vitro at different incubation periods.The pathogen A. solani was grown in 150 ml Erlenmeyer flask containing 25 ml of semi ripe tomato fruit medium.The flasks were sterilized at 15 lb/sq in pressure for 15 min.After sterilization, each flask was inoculated by a 8.0 mm disc taken from the periphery of four day old colony of the pathogen growing on potato dextrose agar medium.The inoculated flasks Chaurasia et al. 249 were incubated for 2, 4, 6, 8, 10 and 12 days under stationary conditions at 28°C.Three replicates were taken in each case.After desired incubation, fungal mat was removed from the medium and the culture fluid was diluted with 35 ml of distilled water.The filtrates thus obtained was centrifuged for 15 min at 10,000 rpm and the supernatant was used as crude enzyme extract.

Production of enzymes in vivo
Semi ripe tomato fruits were used for this purpose.Healthy semi ripe tomato fruits were first surface sterilized with 0.01% mercuric chloride and then washed several times with sterilized distilled water.The surface sterilized fruits were inoculated with A. solani by cavity method (Granger and Hornes, 1924;Chaurasia et al., 2009;Chaurasia and Chaurasia, 2010).The inoculated fruits were kept in sterilized moist chambers having 98-100% relative humidity.These chambers were incubated at 28°C.Healthy surface sterilized fruits were kept as control.After 2, 4, 6, 8, 10 and 12 days of incubation, the infected tissue was taken out with the help of a spatula.Thirty grams of diseased tissue was mixed with 30 ml distilled water and the mixture was homogenized in Waring blender for 10 min.The homogenate thus obtained was centrifused for 20 min at 10,000 rpm.The supernatant was used as enzyme extract.Enzyme extract from uninoculated healthy tissue was also prepared in a similar manner.
At the time of determination of PG, PMG and C X enzyme activity, desired substrate component was taken into the stalk bulb of viscometer.Then, 1.5 ml of freshly prepared enzyme extract was poured into viscometer and then efflux time of the enzyme reaction mixture was determined at the intervals of 0, 20, 40, 60 and 80 min.Efflux time for 8.0 ml of distilled water was also noted in each viscometer.

Determination of percent loss in viscosity
Percent loss in viscosity was calculated with the help of the following formula (Capellini, 1966;Chaurasia et.al. 2013c): where, ET 0 = Efflux time in seconds at zero time/control.ET t = efflux time in seconds at any specific interval of time.ET w = efflux time ET 0 -ET t Percent loss in viscosity  x 100 ET 0 -ET W

Determination of relative enzyme activity (REA)
Values for percent loss in viscosity were determined for 0, 20, 40, 60 and 80 min reaction time.These values were then plotted against the reaction time, thus a curve was obtained and from this curve, the time to bring a 25% loss in viscosity was determined.REA was then calculated using the following formula: 1000 REA = t Where, t = represent the time in min to reach 25% loss in viscosity.Thus:

Production of PG, PMGand Cx enzymes in vitro
The pathogen Alternaria solani was cultured in the semi ripe tomato fruits medium and incubated for 2, 4, 6, 8, 10 and 12 days at 28°C.In obtained culture filtrates, the activity of PG, PMG and Cx enzymes was assayed and results are presented in Table 1 and Figure 1.It is clear that pathogen A. solani was able to produce PG, PMG and Cx enzymes with a short period of incubation, that is, within 2 days.The gradual increase in incubation period up to 6 days, the activity of PG, PMG and Cx enzymes significantly increased in the culture filtrates.The 6 day of incubation period was found to be the best for the maximum production of PG, PMG and Cx enzymes as 80.2 (REA 112.61),40.3 (REA 20.13) and 88.3 (REA 133.33) percent loss in viscosity has been recorded respectively at 80 min of reaction time.After 6 days, further increase in incubation period up to 12 days, has no effect and production of PG, PMG and Cx enzymes declined gradually.Mehta et al. (1974) has reported the maximum production of PG, PMG and Cx enzymes in between 4 to 12 days, in culture filtrates of A. solani and Alternaria tenuis.Mehta and Mehta (1985) also reported maximum PG, PMG and Cx enzyme activity in 6 days old culture of Fusarium oxysporium.The gradual fall in production of PG, PMG and Cx enzymes in long incubation period could be due to the slow inactivation of PG, PMG and Cx enzymes by the appearance of oxidized phenols in the semi ripe tomato fruit medium.The same explanation could hold for the observation of Harter and Weimer (1921) on the production of pectic enzyme by Rhizopus nigricans and Rhizopus artocarpi, in sweet potato broth.Similar observations were made by Balasubramanian and Srivastava (1973).

Production of PG, PMG and Cx enzymes in vivo
Data (Table 2 and Figure 2), indicates the activity of PG, PMG and Cx enzymes in the diseased and healthy tissue of semi ripe tomato fruits, in different incubation periods.From the results, it is evident that PG, PMG and Cx enzymes present in healthy tomato fruits and maximum activity of these enzymes were recorded in 6 days incubition period.After 6 day incubation, with the age, the activity of PG, PMG and Cx enzymes was decreased gradually in healthy tissues.It was also observed that the activity of PG and Cx enzymes has completely disappeared in healthy tomato fruits kept for 12 day incubation.
From the above result, it is concluded that the presence of PG, PMG as well as Cx enzyme in healthy tissues shows their constitutive nature.Comparatively, PG enzyme has been found to be    Ulrich (1958), Mc Cready et al. (1955) has also reported the higher activity of PG enzyme in healthy tissues of various fruits.
In inoculated diseased semi ripe tomato fruit, the activity of PG, PMG and Cx enzymes were found to be higher in comparison with healthy tissue at various given incubation period.Within a short incubation period, that is, within 2 day, activity of PG, PMG and Cx enzymes has been noted in diseased tissues, which increased with increased in incubation period up to 6 days.The 6 days incubation period was found to be the best for the production of PG, PMG and Cx enzymes in diseased tissues, in which 73.04, 65.23 and 0.0 REA were observed respectively.After 6 day incubation, further increase in incubation period had no effect on the production of PG, PMG and Cx enzymes.In 12 day old infected tissues, the Cx activity has completely disappeared which indicates the inactivation of Cx enzyme in a very long incubation period.
Comparatively, the activity of PG and PMG enzymes were found to be always higher than the Cx enzyme in diseased tissues of various incubition periods, which indicated that PG and PMG enzymes played a significant role in pathogenesis of A. solani.To some extent, these results are similar and in agreement with the work of Mehta et al. (1974), Agarwal and Gupta (1978), Hasija and Chawdhury (1979), Sanghi and Rajak (1987) and Sharma (2000).In general, production of PG, PMG and Cx was higher in vitro than in vivo.This could be due to the negligible inactivation of these enzymes by phenols present in the semi ripe tomato fruit medium or perhaps the fungus is forced to secrete the enzymes in large quantities to spread in the semi ripe tomato fruit medium.This situation does not prevail when the fungus is grown in host tissue.The ability of the fungus to secrete PG, PMG and Cx in vitro and in vivo indicates the importance of these enzymes in the pathogenesis of the fungus.

Figure 1 .
Figure 1.Production of PG, PMG and Cx enzymes by A. solani in semi ripe tomato fruit medium at different incubation periods.

Figure 2 .
Figure 2. Production of PG, PMG and Cx enzymes in healthy and diseased semi ripe tomato fruits at different incubation periods.

Table 1 .
Production of PG, PMG and Cx enzymes by A. solani in semi ripe tomato fruit medium at different incubation periods.
in seconds for distilled water.

Table 2 .
Production of PG, PMG and Cx enzymes by A. solani in healthy and diseased semi ripe tomato fruits at different incubation periods.