PECTOLYTIC ACTIVITY OF FUNGI IN STRAWBERRIES cultures of lactic streptococci. J. Dairy Research,, -. WHITEHEAD, H. R., AND COX, G. A. Bacteriophage phenomena in cultures of lactic streptococci. J. Dairy Research,, -. WHITEHEAD, H. R., AND HUNTER, G. J. E. Observations on the activity of bacteriophage in the group of lactic streptococci. J. Path. Bacteriol.,, -. WHITEHEAD, H. R., AND HUNTER, G. J. E. Bacteriophage in cheese manufacture. Contamination from farm equipment. J. Dairy Research,, -. WILKOWSKE, H. H., NELSON, F. E., AND PARMELEE, C. E.. Serological classification of bacteriophages active against lactic streptococci. Appl. Microbiol.,, -. YAKOVLEV, D. A. Bacteriophage of lactic acid streptococci. Microbiology (U.S.S.R.),, - (in English, ). (Original not available; abstracted in Chem. Abs.,,..) The Incidence and Pectolytic Activity of from Michigan Strawberry ' Department of Botany and Plant Pathology and Department of Bacteriology and Public Health, Michigan State College, East Lansing, Michigan Food preservation by freezing has been practiced for many years but its practical application to fresh fruits has developed in about the last twenty years. In order to determine the quality of the small packages of the frozen strawberry fruits a number of standards have been set up for quality control. The use of the Howard mold count method as one of the guides in -quality control dates back to when Dr. Howard introduced this method for use in connection with tomato products. In, Needham and Fellers () applied the Howard mold count to strawberries and black raspberries as an indicator of decay. These first sets of standards were for mold, yeast and bacterial counts. The last two were eventually dropped, and at the present time these frozen fruits may be condemned on the basis of mold counts as an indicator of decomposition and mushiness of the product. Stevens and Wilcox () found that the molds most commonly attacking strawberry fruits were Rhizopus sp., Botrytis sp., Cladosporium sp., Fusarium sp., Mucor sp., and Penicillium sp. The deteriorative action of Rhizopus sp. and Botrytis sp. on strawberries was described in detail by Stevens (). Increasing attention is presently being given to the role of pectolytic enzymes in the separation of tissue in fruits and vegetables and it was felt there was a need to investigate the comparative pectolytic activity of the principal fungi isolated from strawberry fruits. According to Kertesz () polygalacturonase is the enzyme that is Journal Article No. of the Michigan Agricultural Experiment Station and Contribution No. - of the Department of Botany and Plant Pathology. This research was financed by a grant from the Michigan Frozen Food Packers Association, Benton Harbor, Michigan. Received for publication March, capable of splitting off the alpha - glucosidic linkage of the pectin molecule. The enzyme polygalacturonase is commonly produced in varying amounts by many species of fungi depending upon conditions. When this occurs in fruits and vegetables, rupturing of tissue and softening may occur. The pectinesterase content of the substrate has been reported by Jansen and MacDonnell () as one of the limiting factors determining the activity of polygalacturonase. The combined activity of the two enzymes has been included in the term pectinase. None of the microorganisms reported by Smart () in frozen strawberries are known to be pathogenic in humans. The fungi isolated from the fresh washed strawberries were reported as: Alternaria sp., Aspergillus sp., Botrytis sp., Cladosporium sp., Fusarium sp., Monilia sp., Mucor sp., Oidium sp., Penicillium sp., Rhizopus sp., and Stemphylium sp. After years' storage at F, only several species of Penicillium and a couple of yeasts could be isolated and grown from the frozen strawberries. More recently, Anderson () showed that the loss of about per cent of the crop from heavy fruit rot in Illinois during the strawberry season in was primarily due to four organisms: Botrytis cinerea (gray mold), Rhizopus nigricans (leak), Phytophthora cactorum (leathery rot), and Rhizoctonia sp. (black rot). The gray mold which is entirely dependent upon moist field conditions caused the most concern during the ripening period. In the same year, McClintock () reported that Botrytis cinerea was causing most of the strawberry fruit rot in Indiana. During the summer of, Alexopoulos and Cation () first reported Dendrophoma obscurans as the Downloaded from http://aem.asm.org/ on August, by guest
cause of a stem end rot in. per cent of the Robinson strawberries collected in Michigan. Other fungi found in the fruits were reported as Botrytis cinerea, Pezizella lythri, Phytophthora cactorum, Rhizopus nigricans, Rhizopus stolonifera, and Penicillium sp. OBJECTIVES It has been long recognized that some fungi cause rot in strawberry fruits. However, it has not been shown that mold counts in these fruits, as determined by the official Howard mold count method which is given in the Ofjicial Methods of the Association of Agricultural Chemists (), represent only rot-producing organisms. It was thought that many harnless saprophytes which are not active causes of rotting may be included in the present mold count method. This has been shown to be the case in black raspberries by White and Fabian (). It was demonstrated that two of the commonly found fungi in black raspberries, Alternaria humicola, and Cladosporium sp. produced relatively little or no pectolytic activity while Pullularia pullulans, Fusarium sp., Botrytis cinerea, and Penicillium sp. showed greater activity. Pandhi () in a scientific and technical survey on the softening and breakdown of vegetables and fruits states that, "A knowledge of the moulds that commonly attack strawberries, of the production of the pectic enzymes by these moulds and the elucidation of the mechanism of the action of the PG-complex on pectic acid will also help a great deal in the solution of the problem." The purpose of this investigation was to determine:. The types of fungi found in various stages of TABLE. Alternaria sp... Aspergillus niger... Botrytis cinerea... Pezizella lythri... Helminthosporium Sp... Hormodendron sp... Mucor sp... Mycelia sterilia... Penicillium sp... Pullularia pullulans... Rhizopus nigricans... Torula sp... Yeasts... Trichoderma sp... Epicoccum sp... growth of the strawberry fruits around the Benton Harbor, Michigan, area.. The frequency of the different types of fungi on or in the strawberry fruits.. Mold counts as influenced by stage of ripeness, temperature, humidity, and storage time.. The pectin destroying activity of the principal types of fungi encountered. METHODS Samples of fresh strawberries were taken at different stages of maturity, which included green fruits and ripe fruits, some of which had various types of rot developing. Some of these samples of fruits were treated on the surface by immersion for minute in a solution containing two parts of sodium hypochlorite and one part of per cent ethyl alcohol. Plugs were cut out rrom the treated fruits and placed on both Bacto W. L. nutrient medium and potato dextrose agar. Likewise, plugs were cut out from the surface of untreated strawberries to get representative types of fungi or fungus spores that normally were present. Periodically, the growth of the fungi was examined and as the reproductive structures developed, the organisms were identified. Isolations were made for the pectolytic studies. A total of plugs were studied for types of fungi that normally were found on and in green and ripe strawberries during the season in Michigan. The varieties of strawberries sampled included Premier, Early Red June, Robinson, Catskill, Sparkle, and a number of hybrids. The first three varieties were sampled June, (table ). This was during a cool, Incidence of fungi on the surface and subsurface of three varieties of green strawberries grown in nine different fields in Michigan Variety Premier Early Red June Robinson No. of isolates from No. of isolates from No. of isolates from plugs in surface plugs in surface plugs in surface I, I_ Surface- Totals. Downloaded from http://aem.asm.org/ on August, by guest
PECTOLYTIC ACTIVITY OF FUNGI IN STRAWBERRIES TABLE. Alternaria sp... Aspergillus niger... Aspergillus sp... Botrytis cinerea... Epicoccum sp... Fusarium sp... Pezizella lythri... Hortiodendron sp... Mucor sp... Mycelia sterilia... Penicillium sp... Pullularia pullulans... Rhizopus nigricans... Rhodotorula sp... Torula sp... sp... Trichoderma Yeasts... Monilia sp... Incidence of fungi on the surface and subsurface of three varieties of ripe strawberries grown in nine different fields in Michigan Variety Premier Early Red June Robinson No. of isolates from No. of isolates from No. of isolates from plugs in surface plugs in surface plugs in surface Totals... TABLE. Incidence of fungi on the surface of several varieties of ripe strawberries grown in eight different fields in Michigan Number of Isolates from Plugs in Variety Hybrids, lu Total Premier Early Red Robinson Catskills Sparkle June Alternaria sp...... Aspergillus niger. Aspergillus sp. Botrytis cinerea. Epicoccum sp. Pezizella lythri. Helminthosporium sp. Hormodendron sp. Mucor sp. Mycelia sterilia. Penicillium sp. Pullularia pullulans. Rhizopus nigricans. Rhizopus sp. Rhodotorula sp. Trichodernta sp... Trichoderma album. Yeasts... Monilia sp. Dendrophoma obscurans... Total. Surface- Downloaded from http://aem.asm.org/ on August, by guest wet period with intermittent showers and temperatures reaching a high of about F. Samples of the same three varieties were taken week later, June, (table ), a partly cloudy day during a dry period when the temperature reached a high of about F in the afternoon. A third series of samples including all of the above named varieties was taken on June, (table ) after a dry period of nearly days. At this time there
were iintermittent showers occurring and the temperature reached about F. Firm, ripe strawberries were inoculated with spores from cultures from fungi most frequently isolated in this study, which were Botrytis cinerea, Rhizopus nigricans, Aspergillus sp., Pullularia pullulans, Trichoderma sp., Penicillium sp., Hormodendron sp., and Alternaria sp. The fruits were then placed at - C in a relative humidity of to per cent. Visual observations of the appearance and firmness of the fruits as well as ph values were recorded at the periodic intervals. Samples of these inoculated strawberries were extracted by means of a laboratory pulper at hours, hours, hours and hours after they had been inoculated. The extracts were adjusted to a ph of., after which per cent salt was added and the samples filtered through cheesecloth. The extracts were then brought to a standard volume and stored under toluene at C until examined. For testing, ml of the extract was added at C for hours. Heat inactivated extracts provided controls. Measurement of the decrease in the viscosity of a test pectin solution was employed to provide an indication of the pectinase complex. The method was the same as that employed in a similar study by White and Fabian (), except an -hour incubation period was used. RESULTS A total of fungi were isolated from samples of green and ripe strawberry fruits from approximately miles square area around Benton Harbor, Michigan. The total number of fungi isolated from the surface of the fruit was, while the total number found below the surface of the fruit was. There was a marked increase in the total number isolated from the subsurface of the ripe fruits, increasing from isolates in green fruits to in ripe fruits. This would be expected, as ripe strawberries are more susceptible to fungus growth. A total of fungi was isolated from the untreated surface of the green fruits while a total of fungi was identified from the untreated surface of ripe fruits. The incidence of fungi on the surface and subsurface in green fruits of the three varieties, Premier, Early Red June, and Robinson is shown in table. Some of the fungi found growing below the surface of the green fruits have been previously reported as developing in green fruits-notably Botrytis cinerea, and Pezizella lythri. Others that were isolated a number of times from green fruits and which have not previously been reported were: Rhizopus nigricans, yeasts, Mycelia sterilia, Hormodendron sp., and Penicillium sp. However, the number of isolates of fungi showed an increase in the subsurface of ripe fruits as compared to greenl fruits as shown in table. These additional genera of fungi were also present in some of the ripe fruit, namely, Mucor sp., Aspergillus sp., and Epicoccum sp. As would be expected, the total number of fungi found on the surface of fruits is much greater than that below the surface of green or ripe strawberry fruits. There is no apparent difference between the kind of fungi on the surface of green and ripe fruits. The kind of spores on either of these surfaces should be similar since most of the fungus spores are air-borne. Table lists only fungi isolated from the surface of the ripe fruits. These samples were taken towvard the end of the harvest period, June,, after about days of dry weather. Essentially the same types of fungi could be found after this dry period although a few fungi showed marked increases in numbers of spores on the surface of fruits. These were Altermaria sp., Botrytis cinerea, Aspergillus sp., Pezizella lythri and Rhizopus nigricans. These organisms are very likely to be growing in rotted fruits which results in more spores being produced and a greater per cent of these genera would make up the total number of spores on the surface of fruits in the area where samples were taken. In some cases, for example, Hormodendron sp. and Fusarium sp., a drop in the frequency of isolation occurred during dry weather. These organisms are commonly isolated from soil, but during a dry period there is less soil likely to be on the fruits, because soil particles are not splashed onto the surface by water droplets. Therefore the amount of spore inoculum would be reduced during a dry period. Although some of these organisms occurred more frequently on the surface of normal green and ripe fruits, there would be no marked increase in mold counts during dry weather, since spore germination usually does not occur readily under these conditions. In wet weather the reverse would be true. It is interesting to note that in the Robinson variety relatively few fungi were isolated from the subsurface of the green or ripe fruits. However, this variety matures a little later than the Premier or Early Red June, and a dryer atmospheric and soil condition prevailed at this time when the green fruits were enlarging and ripening which would result in less favorable conditions for the germination of the fungus spores. This may account for the very low number of fungi isolated from the subsurface of these fruits. During the entire strawberry season, the mold counts were generally low since dry conditions prevailed. Frequently, few or no fungi were found in the fruits when mold counts were made during the dry part of the season. A similar situation was reported at the Cornell Agricultural Experiment Station by Steinkraus (). The samples of strawberries as well as samples of raspberries showed low mold counts. He showed that washing and sorting procedures used by processing plants were not very effective in lowering mold counts. The fact that length of time between pickings showed less effect on mold Downloaded from http://aem.asm.org/ on August, by guest
PECTOLYTIC ACTIVITY OF FUNGI IN STRAWBERRIES counts than did rainfall emphasizes the difficulties involved in maintaining low mold counts in humid weather. The list below is a compilation of the total number, obtained in this study, of the most commonly isolated fungi on the surface of green and ripe strawberry fruits: Genus No. of Isolates Hormodendron sp... Yeasts... Rhizopus sp... Alternaria sp... Pullularia sp... Penicillium sp... Botrytis sp... Pezizella sp... Aspergillus sp... Trichoderma sp... Mucor sp... It is well known that Rhizopus nigricans and Botrytis cinerea are among the genera more commonily involved in the breakdown of strawberry fruits. However, the significance of the various other fungi found on the strawberry fruits has not been determined. The testing for pectinase activity of these various fungi has shown significant differences in their rate of activity and in an important aid in determining the significance of these fungi in strawberry fruits (figure ). The greater pectolytic activity of some of the more important fungi on the above list does not necessarily correlate with the frequency of isolation. The individual genera of fungi that were inoculated into strawberry fruits showed little difference in the relative pectinase activity after hours. No visible microscopic growth was evident at this time in any of the inoculated fruits. However, after hours the uninoculated control sample showed no visible growth while the fruits inoculated with Rhizopus nigricans had greater visible growth than did those inoculated with Alternaria sp., Trichoderma sp., and Penicillium sp. The typical leak symptoms were evident in the Rhizopus-inoculated samples while none of the others showed this condition. Less deteriorative activity was evident from visual observations. While all of the samples including the control were shown to contain a lower amount of pectinase activity in the berries immediately after inoculation in comparison to the results after hours, only Rhizopus nigricans produced a significantly higher titer in this short period of time. All of these higher titers are associated with a slight drop in the ph value of the strawberries. This was even more evident after hours. The ph values for the samples were approximately. at the beginning of the experiment and at the end of hours the ph values were approximately. for all of the samples except Rhizopus nigricans BADLY DECOMPOSED BERRIES Aspergillus sp. otrytis sp. - l-rhiz.opus Sp.\ I I OTHER BERRIES Alt-rnaria sp. PuIluIari@ sp. - Hormod*ndrum sp. Pnicillium sp.\ Trichoderma Sp. MOLD CONTACT TIME, HOURS K I I I- I ^ FIG.. The pectolytic activity in strawberries of seven representative molds as measured by the viscosity method. which had a drop in the ph value from. to.. Figure shows the relative per cent drop in viscosity of a per cent test pectin solution produced by extracts from strawberries inoculated with the seven most commonly found molds. After hours, all samples showed varying degrees of deterioration. The fruits inoculated with Botrytis cinera, Rhizopus nigricans and Aspergillus sp. were badly decomposed. The pectinase activity of these extracts was similarly much greater than that noted for the samples inoculated with other fungi. All samples, however, showed a consistently higher titer of pectinase activity after than at hours. It is recognized that there are limitations to the viscosity method of measuring pectinase activity as shown by Kertesz (). However, the application of this method does show the pronounced variations which occur in the production of pectolytic enzymes by fungi. Two organisms, Alternaria humicola and Cladosporium sp. were shown previously by White and Fabian () to produce little or no pectolytic activity when grown in black raspberry extracts. These variations in pectinase activity by fungi emphasize further that the presence of a mold in a food product may not necessarily be associated with the pectolytic breakdowni of that product. In, other studies by Proskuriakov and Ossipov () have also shown that variation in the production of pectolytic enzymes may be due to individual strains in the various species of fungi. Thus, in the microscopic examination of a product, when the number of positive fields for fungi is found to increase toward per cent, as determined by the official Howard mold count method, the observation may not necessarily be taken as an exact index of the actual amount of decomposition of a food product because some of the fungi found in strawberry fruits are not important in pectolytic breakdown. Downloaded from http://aem.asm.org/ on August, by guest
SUMMARY A total of fungi, from the surface and from the subsurface, were isolated from samples of green and ripe strawberry fruits in the vicinity of Benton Harbor, Michigan, during the season. A total of isolates of fungi were made from the subsurface of green fruits. These were Botrytis cinerea, Pezizella lythri, Rhizopus nigricans, yeasts, Mycelia sterilia, Hormodendron sp., Penicillium sp., Alternaria sp., Helminthosporium sp., and Pullularia pullulans. Additional genera, Mucor sp., Aspergillus sp., Epicoccum sp., and Monilia sp. were isolated from the subsurface of ripe fruits, -making a total of isolated from the subsurface of ripe fruits. Of the fungi isolated from the surface of green and ripe strawberry fruits, the ones found most commonly, listed in decreasing frequency of isolation, were: Hormodendron sp., yeasts, Rhizopus nigricans, Alternaria sp., Pullularia pullulans, Penicillium sp., Botrytis cinerea, Pezizella lythri, Aspergillus sp., Trichoderma sp., and Mucor sp. The mold counts during the season in Michigan were generally very low due to dry weather conditions. When the various genera of fungi were inoculated into fresh strawberry fruits, strains of Botrytis cinerea, Rhizopus nigricans and Aspergillus sp., were shown to produce macroscopically greater degradation than Pullularia pullulans, Trichoderma sp., Penicillium sp., Hormodendron sp., and Alternaria sp. The apparent degradation was consistently found to be associated with a greater pectinase content in the affected strawberries. The variation obtained in pectinase activity by fungi emphasizes further that the amount of mold in a food product as determined by the mold count method may not necessarily be associated with the pectolytic breakdown of that product. REFERENCES ALEXOPOULOS, C. J., AND CATION, D. Stem end rot of strawberries. Phytopathology, -. ANDERSON, H. W. Strawberry fruit rots and their control. Trans. Illinois State Hort. Soc., -. BEAVEN, G. H., AND JONES, V. K. N. Molecular structure of pectic acid. Chemistry and Industry,,. JANSEN, F. E., AND MACDONNELL, L. R. Influence of methoxyl content of pectic substances on the action of polygalacturonase. Arch. Biochem.,, -. KERTESZ, Z. I. The pectic substances. Interscience Publishers, New York. MCCLINTOCK, J. A. Spraying for strawberry fruit rots in. Housier Hort.,, -. NEEDHAM, G. H., AND FELLERS, C. H.. An application of the Howard method to the detection of spoilage in berry products. J. Assoc. Offic. Agr. Chemists,, (), -. Official Methods of Analysis of the Association of Official Agricultural Chemists, th ed. XV. Washington, D. C. PANDHI, P. N. The softening and breakdown of vegetables in brine and of fruits-in solutions of sulphur dioxide. Scientific and Technical Surveys No., December. The British Food Manufacturing Industries Research Association, Leatherhead, Surrey, Randalls Road, England. PROSKURIAKOV, N. J., AND OssiPov, F. M. Biokhimiya, (PHAFF, H. J.). The production of exocellular pectic enzymes bvypenicillium chrysogenum. I. On the formation and adaptive nature of polygalacturonase and pectinesterase. Arch. of Biochem., (), -. SMART, H. F. Microorganisms surviving the storage of frozen-pack fruits and vegetables. Phytopathology,, -. STEINKRAUS, K. H. Mold count index to fruit quality. The Canning Trade,,. STEVENS, N. E., AND WILCOX, R. B. Further studies of the rots of strawberry fruits. U. S. Dept. Agr. Bull.,. pages. STEVENS, N. E.. Keeping qualities of strawberries in relation to their temperature when picked. Phytopathology,, -. WHITE, L. S., AND FABIAN, F. W. The pectolytic activity of molds isolated from black raspberries. Appl. Microbiol.,, -. Downloaded from http://aem.asm.org/ on August, by guest