J. Sci. Food Agric. 1979, 30, 687-691

The Involvement of Fungi in the Breakdown of Sulphited Straw berries

Colin Dennis and Jane E. Harris

ARC Food Research Institute, Colney Lane, Norwich NR4 7UA

(Manuscripf received 24 October 1978)

The presence of strawberries infected with Mucor piriformis, Rhizopus sexualis, or R. stoloxlifer in a sample of sulphited whole berries caused complete breakdown of the whole sample of fruit. As little as 1-2% of infected tissue for each of the three species consistently caused breakdown while with R. sexualis even 0.1 % resulted in some softening and breakdown of the fruit. Similar results were obtained when culture filtrates possessing pectolytic activity, of each of the above fungi, were added to sulphited fruit. In addition culture filtrates of other pectolytic fungi associa- ted with strawberries, Aureobasidium pullulans, Trichosporon pullulans and Crypto- coccus albidus var. albidus were also shown to cause breakdown. Jt is suggested that the disintegration of the fruit is due to fungal pectolytic enzymes, most probably polygalacturonases. The addition of berries infected with Botrytis cinerea or culture filtrates of this fungus, possessing pectolytic activity, to sulphited healthy fruit did not cause any softening or breakdown.

1. Introduction

In order to ensure a continuous supply of fruit throughout the year for jam manufacture, quantities of strawberries are stored frozen or preserved in sulphite liquor prior to manufacture. Approxi- mately 70 % of the eight to nine thousand tons of strawberries used annually for jam in the UK is sulphited. When properly controlled, this is a cheap and efficient method of storage providing an ideal raw material for the production of good retail jam, which should contain whole fruit or at least recognisable pieces of fruit.

For many years in the UK there have been sporadic cases of disintegration of sulphited strawberries.a Over recent seasons this problem has become more serious and there have been indications of a greater incidence of breakdown of fruit from certain fruit growing areas compared to others.

Previous work1 on the cause of disintegration of such fruit in England and Holland had indicated that the breakdown of sulphited fruit could be due to the activity of pectolytic enzymes but no proof of enzymic breakdown has been reported. Dutch workers2 had suggested that such enzymes were of fruit origin, whereas Dakin and Tampion1 had implicated fungal enzymes as the cause of breakdown. The presence of strawberries infected with the Zygoniycetes, R. sexualis, R. stolonifer or Mucor spp. resulted in the breakdown of sulphited fruit, with R . stolonifer being the most active species, while the presence of berries infected with B. cinerea did not affect the texture of the fruit.l In an analogous situation with sulphited cherries Weigand3 and Steele and Yang4 showed a cor- relation between breakdown and the presence of pectolytic enzymes of certain fungi.

Sulphited strawberries are usually referred to by growers and processors as ‘pulp’; a term which is used for all qualities, from whole fruit to completely disintegrated fruit.

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688 C. Dennis and J. H. Harris

Recent work53 has also shown that the main spoilage organisms of strawberries grown in the UK are B. cinerea and Mucor pirijiormisa with Rhizopus species only accounting for a small pro- portion of the spoilage. Mucor hiemalis and M. racemosus have also been isolated from rots but mostly developed as secondary spoilage fungi.

The present series of papers re-examines the cause and mechanism of breakdown of sulphited strawberries and also assesses the relative importance of different spoilage fungi in the breakdown of commercial samples of fruit from different growing areas. The first paper is concerned with the potential of fungi associated with strawberries to cause disintegration of sulphited fruit.

Since Dakin and Tampion1 did not specify which species of Mucor they used and also because of the predominance of Mucor over Rhizopus in the spoilage of strawberries it was necessary to investi- gate the ability of different Mucor species to cause breakdown. Further since Dakin and Tampion had only tested one strain of B. cinerea, a fungus which is very variable in its physiological pro- perties,7 more strains, freshly isolated from strawberries, were tested. In addition other pectolytic fungi known to associated with strawberriess were included in the study.

2. Experimental 2.1. Culture Most of the isolates of B. cinerea, M. piriformis, M. hiemalis, M. racemosus, R. sexualis and R . sfolonifer were isolated from rots of strawberries while the cultures of Aureobasidium pullulans, Trichosporon pullulans and Cryptococcus albidus var. albidus were obtained from the surface of strawberry fruits. One isolate of B. cinerea was from a gooseberry fruit and one isolate of each of M. piriformis and R . stolonifer was obtained from raspberries. Cultures of B. cinerea, Mucor and Rhizopus species were maintained on Difco Bacto potato dextrose agar (PDA) while A. pullulans and the yeasts were maintained on Difco Bacto YM agar (YMA).

2.2. Preparation of infected fruit Strawberries of the variety Cambridge Favourite were grown in pots in the glasshouse or in field plots at the Food Research Institute. The ripe fruits were carefully harvested without the calyx and inoculated at the calyx scar by means of a mycelial disc cut from the leading edge of a culture growing on PDA (B. cinerea, Mucor spp. and R . stolonifer) or YMA pH 4.8 ( R . sexualis) at 20°C The inoculated fruit was incubated in a moist chamber at 15°C for about 2 days when the fruit was half to two-thirds rotted.

2.3. Preparation of culture filtrates Asexual spores were harvested by adding 10 ml 0.1 % Tween 80 to cultures grown on RNAAlO (B. cinerea), PDA (M. piriformis and R . stolonifer) or YMA ( R . sexualis) slopes at 20°C for 3-6 days, vigorously shaking the slopes and rubbing the culture with a sterile needle. Each spore suspension was filtered through two layers of sterile muslin to remove mycelial fragments. The filtered spores were centrifuged and washed twice with 10 ml sterile glass distilled water (SGDW) before adjusting the concentration to approximately 105 spores ml-I. Cells of A. pullulans, T. pullulans and C. albidus var. albidus were harvested from cultures grown on YMA slopes at 20°C for 3 days by adding 10 ml SGDW and washed twice with 10 ml SGDW before adjusting the cell suspension to approximately 0.1 mg ml-1 dry weight.

Conical flasks (100 ml) containing 25 ml of a polypectate medium pH 5.0 (Sunkist Na poly- pectate, 10 g litre-I; ammonium tartrate, 15 g litre-I; KH2P04, 2 g litre-l; MgS04.7H20, 0.5 g litre-]) were inoculated with 1 ml of a spore suspension or 0.3 ml of a cell suspension. The inocu- lated flasks were incubated on an orbital shaker at 20°C. After 3 days’ incubation the contents of

a In all previous publications from this laboratory the name M . mucedo has been used as a result of using the key of Zycha et a17 and on the advice of the Commonwealth Mycological Institute. A recent re-examination of the strains resulting from Dr M. Schippers revision of the genus places our isolates in M . piriformis. This has been confirmed by mating experiments with type strains.

Fungal breakdown in sulphited strawberries 689

the flasks were filtered through Whatman No. 3 filter paper prior to centrifuging at 12 000 rev min-1 for 10 min at 2°C to give a cell-free culture filtrate for use in sulphiting experiments.

2.4. Sulphiting procedure 2.4.1. Infected fruit experiments Healthy ripe strawberries without the calyx (250 g), of the variety Cambridge Favourite, or a mix- ture of healthy fruit and infected fruit or tissue, were placed in 454 g Kilner jars. To each jar 50 ml of a 2% solution of sulphur dioxide saturated with slaked lime (bisulphite of lime) was added. This gave a final concentration of between 2000 and 3000 parts sulphur dioxide as determined by the Monier-Williams distillation method11 and a final pH of approximately 3.0 in the sulphite liquor. The jars were shaken to ensure thorough mixing of the fruit and liquor prior to storage at 15°C.

In initial experiments 25 % of fruit infected with each of the spoilage fungi was included in samples of healthy fruit and sulphited as described below. In other experiments only the rotted tissue from the infected fruit was used. For each species the rotted tissue was removed, pooled and then 0.1, 0.5, 1.0, 2 or 5 % added to healthy fruit before sulphiting.

Each treatment was duplicated and a similar set of duplicate jars containing 5 % infected tissue, which had been boiled in 4 g amounts for 1 h to destroy any pectolytic enzymes, were also set up.

2.4.2. Culture filtrate experiments

Each culture filtrate (25 ml) was mixed with 25 ml of a 4% solution of sulphur dioxide saturated with lime and then added to 250 g of healthy ripe strawberries in 454 g Kilner jars as described above. Similar jars containing 25 ml of boiled (1 h) culture filtrates were set up and all treatments were done in duplicate.

2.5. Assay for pectolytic activity All samples of culture filtrates and sulphite liquors were dialysed overnight at 2.5"C against deion- ised water prior to assaying. Pectolytic activity was assayed using the agar plate technique described by Archer12 except that salicylanilide was replaced by ethyl mercurithiosalicylate (thimerosal) to ensure no microbial growth on the plates during the assay. Pectinol 1 0 ~ concentrate (Rohm and Haas) at 5 mg ml-1 was used as a standard.

3. Results 3.1. Ability of spoilage fungi to cause breakdown When 25% of half to two-thirds rotted berries were included in sulphite liquor with healthy fruit, complete disintegration of all of the berries occurred within 6-8 weeks for fruit infected with strains of M. piriformis, R. sexuulis and R. stolonifer. No breakdown occurred with fruit artificially infected with any strains of B. cinereu or with any of the samples of naturally infected fruit collected from a strawberry plantation (Table 1). Mucor hiemulis and M. racernosus were not able to infect the strawberry fruits when inoculated on the calyx scar before other spoilage fungi invaded the berries and therefore no further tests were done on these two species.

3.2. Ability of culture filtrates to cause breakdown The addition of culture filtrates from each of the spoilage fungi to sulphited fruit confirmed the above observations since only filtrates from the Mucor and Rhizopus species caused disintegration of the fruit. The culture filtrates of B. cinereu, although possessing the same level of initial pectolytic activity as assayed by the agar plate method, had no obvious effect on the texture of the fruit.

Tests with culture filtrates of other pectolytic fungi showed that eleven of the twelve strains of A. pullulans and each strain of T. pullulans and C. albidus var. albidus tested were capable of com- pletely distintegrating the fruit over a 6-8 week period (Table 1).

690 C. Dennis and J. H. Harris

Table 1. Ability of fungi to cause breakdown of sulphited strawberries

Species No. of No. of strains

Additions strains tested causing breakdown

Phycomytes M . piriformis Infected fruit 15

Culture filtrate 1 R. sexualis Infected fruit 6

Culture filtrate 1 R. slolonifer Infected fruit 6

Culture filtrate 1

15 1 6 I 6 1

Fungi Imperfecti B. cinerea Infected fruit 6 0

Naturally infected fruit 10 samples 0 Culture filtrate 2 0

A . pullulans Culture filtrate 12 11 T. pullulans Culture filtrate 2 2 C. albidus var. albidus Culture filtrate 1 1

In all cases where filtrates were active in causing breakdown of the fruit, this ability together with the pectolytic activity was destroyed by boiling the filtrate prior to its addition to the fruit.

3.3. Amount of infected tissue required to cause breakdown Further experiments with single strains of M. piriformis, R . sexualis and R. stolonifer showed that as little as 1-2% infected tissue consistently caused breakdown of the 250 g samples for all three species. In the case of R . sexualis even 0.1 % resulted in some softening and disintegration of the fruit (Figure 1).

Figure 1. The breakdown of sulphited strawberries (var. Cambridge Favourite) caused by different amounts of tissue infected with R. sexualis.

Assays carried out immediately after sulphiting of the infected tissue with healthy fruit, detected pectolytic activity in the liquors only at the 5 % level of infected tissue for M . piviformis and R. stolonifer but down to 1 % infected tissue for R . sexualis. Detectable pectolytic activity in the liquors disappeared within 7 days of sulphiting for the former species but persisted for 4, 5 and 20 weeks respectively at the I , 2 and 5 % level of infected tissue of R . sexualis.

Fungal breakdown in sulphited strawberries 69 1

4. Discussion

Thus using a more extensive range of strains of the fungi isolated from strawberry fruits, Dakin and Tampion’s1 conclusions on the ability of Rhizopus species and inability of B. cinerea to cause breakdown have been confirmed. Despite B. cineren being a fungus with very variable physio- logical properties, it is considered that sufficient strains have been tested to show that this fungus does not play a role in the breakdown of sulphited fruit. Contrary to the previous report, R . sexualis appears to be the most active Zygomycete; the addition of extremely low amounts of infected tissue causing disintegration of the fruit.

The present experiments have also shown that M. piriformis is likely to be the Mucor species involved in the breakdown of fruit. In addition A.pullulans and two species of yeast were also shown to possess a similar potential. Although it is unlikely that these latter fungi will develop to any extent on the fruit prior to harvest, development may occur on harvested fruit which is left at ambient temperature for several hours before sulphiting. This is especially true for A . puZluZans and C. albidus var. albidus which are usually present in relatively high numbers on most samples of straw- berriesg

The destruction of pectolytic activity in culture filtrates and infected tissue by boiling, with a corresponding loss of ability to cause breakdown, provides evidence that pectolytic enzymes of fungal origin are the primary cause of breakdown (cf. Staden2). This is analogous to softening of sulphited cherries4 except that different fungal species are involved. In the present experiments very low levels of infected tissue were shown to cause breakdown, although no pectolytic activity could be detected in the liquors by the agar plate method. However, this may be a reflection of the sensitivity of the assay method (see Harris and Dennisl3) rather than the absence of the appropriate enzyme. The evidence from the boiled treatments together with the report by Sommer e l that very small amounts of juice from apricots infected with Rhizopus spp. caused softening of canned apricots over a 7.5-month storage period, still suggests the involvement of pectolytic enzymes.

In the present experiments no attempt has been made to determine which kind of pectolytic enzymes were present in the infected tissue or culture filtrates. The agar plate method used was designed to assay for polygalacturonase activity since this is the only pectic depolymerising enzyme reported to be produced by Zygomycetes.15 Evidence for the involvement of specific pectolytic enzymes in the breakdown of sulphited strawberries is reported by Archer.lG

Acknowledgement The authors thank Mr A. Shirsat for technical assistance.

References 1. Dakin, J. C.; Tampion, J . J . Food Technol. 1968, 3, 39. 2. Staden, 0. L. Annu. Rep. of the Sprenger Institute Wageningen, Netherlands, 1964, p. 42. 3. Weigand, E. H. Proc. Oregon Horlic. SOC. 1957, 47-49. 4. Steele, W. F.; Yang, H. Y . Food Technol. 1960, 14, 121. 5. Dennis, C. Ann. Appl. Biol. 1975, 81, 227. 6. Dennis, C.; Davis, R. P. Proc. 1977 British Crop Protection ConJ-Pest and Diseases 1977, 1, 203. 7. Zycha, H.; Siepmann, R . ; Linnemann, G. Mucorales Verlag von J. Cramer, Germany, 1969. 8. Jarvis, W. R. Botrytitiia and Botrytis species: taxonomy, physiology, and pathogenicity Monograph No. 15,

Canada Dept. Agric., 1977. 9. Dennis, C. In Microbiology of Aerial PIant Surfaces (Dickinson, C. M.; Preece, T. F., Eds.) Academic Press,

1976, p. 419. 10. Geeson, J. D. Ann. Appl. Biol. 1978, 90, 59. 11 . Monier-Williams. G. W. Analyst (London) 1927, 32, 415. 12. Archer, S. A. Physiological aspects of brown rot of apple Ph.D. Thesis, University of Bristol, 1972. 13. Harris, J. E.; Dennis, C. J . Sci. Food Agric. 1979, 30, 704. 14. Sommer, N. F.; Buchanan, J. R. ; Fortlage, R. J.; Gordon Mitchell, F. Calif: Agric. 1977, 31, 16. 15. Rombouts, F. M.; Pilnik, W. Crit. Rev. Food Technol. 1972, 3, I. 16. Archer. S. A. J. Sci. Food Apric. 1979, 30, 692.