Use of the Natural Plant Products, Hinokitiol, to Extend Shelf-life of Peaches1

Can. Inst. Sci. Technol. J. Vol. 24, No. 5, pp. 273-277,

1991

RESEARCH

Use of the Natural Plant Products, Hinokitiol, to Extend Shelf-life of Peaches l P .L. Sholberg Agriculture Canada Research Station, Summerland, British Columbia VOH lZ0 and

B.N. Shimizu Shimizu Bioresearch Ltd., #113 3705 Willingdon Avenue, Burnaby, British Columbia V5G 3H3

Abstract Hinokitiol which is an extract of steam distillation of the root or trunk of Japanese cypress was tested as a control of postharvest decay pathogens of harvested peaches. The effective dose of hinokitiol that reduced fungal spore germination 50070 (ED 50) was 30.0, 14.7 and 18.1 Itg/mL, respectively, for Botrytis cinerea, Monilinia fructicola and Rhizopus oryzae. Hinokitiol killed M. fructicola spores at 20 Itg/mL and inhibited mycelial growth rate 50070 when compared to growth on a medium free of hinokitiol at 14.6, 17.3 and 39.7 Itg/mL for two isolates of M. fructicola and B. cinerea, respectively. Hinokitiol prevented decay of commercially harvested peaches in which 41.1070 of the untreated fruit developed brown rot.

Resume L'hinokitiol est un extrait des racines ou du tronc du cypres japonais, obtenu par distillation a la vapeur. 11 fut essaye pour le contrale des pathogenes responsables de la decomposition postrecolte des peches. La dose efficace d'hinokitiol qui reduisit de 50070 (ED50) la germination des spores fongiques fut 30.0, 14.7 et 18.1 Itg/mL, respectivement, pour Botrytis cinerea, Monilinia fructicola et Rhizopus oryzae. L'hinokitiol detruisit les spores de M. fructicola a la dose de 20 Itg/mL et inhiba de 50070 letaux de croissance du mycelium, par comparaison ala croissance sur un milieu depourvu d'hinokitiol, aux concentrations de 14.6, 17.3 et 39.7 Itg/mL, respectivement, pour deux isolats de M. fructicola et pour B. cinera. La decomposition des peches recoltees commercialement fut empechee par l'hinokitiol, alors que 41.1070 des fruits non traites developperent la pourriture brune.

Introduction Shelf-life of peach fruit can be reduced to a few days after harvesting if decay-causing fungi are not controlled. American brown rot caused by Monilinia fructicola will decay ripe fruit in 2 d under warm wet weather conditions (Jones and Sutton, 1984). Rhizopus rot caused by Rhizopus spp. often 1Contribution Number 774.

Copyright ©

1991

accompanies brown rot on peaches and can be particularly damaging in peaches held by canneries for ripening (Meheriuk and McPhee, 1984). Decay from Penicillium spp. and Eotrytis cinerea occurs only in fruit held in cold storage (0-4°C) for extended periods (Wilson and Ogawa, 1979). Generally peaches shipped to distant markets or held for canning are treated with 1200 Jlg/mL dichloran for control of Rhizopus spp. and 250 Jlg/mL benomyl to control Monilinia fructicola and Penicillium spp. These fungicides have been very effective in providing postharvest disease control on peaches (Wilson and Ogawa, 1979). Fungicides are essential if peaches are to be protected from infection by these pathogens. Recently fungicides have come under public scrutiny and are deemed undesirable because of the possible residues they may leave on the fruit (Wilson and Wisniewski, 1989). Furthermore, many postharvest pathogens have developed resistance to them (Eckert, 1988). Alternatives to fungicides are necessary if postharvest diseases are to be adequately controlled in the future. Natural, plant products with low mammalian toxicity are an alternative to synthetically produced fungicides. One such product, benzaldehyde, totally inhibited spore germination of Eotrytis cinerea at 25 JlL/L and germination of M. fructicola at 125 JlL/L (Wilson et al., 1987). Treatment of grapes· for 24 h with 5000 ppm acetaldehyde vapor, which is a natural biological substance found in all kinds of plant and animal tissues, reduced decay by 92070 as compared to the control (Avissar et al., 1989). Apparently natural plant products can be very effective in the control of postharvest decays. Hinokitiol extract, obtained by the steam distillation of the root of an Hiba

Canadian Institute of Food Science and Technology

273

arborvitae, a species of Hinoki (Japanese cypress) and purification of the given oily portion, showed promise as a general antibiotic against bacterial and fungal isolates of several genera in Japanese tests (Communication from Osaka Branch of National Hygenic Laboratory, Osaka, Japan, 1987). The object of this study was to determine the effectiveness of hinokitiol against decay causing fungi of peaches and to test this material for extending shelf-life of harvested peaches.

Materials and Methods

Fungistatic Effect of Hinokitiol on Spore Germination Hinokitiol formulated as a white powder of 10070 hinokitiol in cyclodextrin (H-D) was supplied by Shimizu Bioresearch Ltd., Burnaby, British Columbia. Ten to forty J.tg/mL active ingredient of the powder was added to Potato Dextrose Agar (PDA) (Difco, Detroit, MI) after autoclaving for 15 min at 120°C and cooling to 50°C. Five fungal isolates were tested. Spores were harvested from 9-day-old cultures of Botrytis cinerea Pers. Fr. grown under black light at 22°C, two Monilinia jructicola (Wint.) Honey isolates grown on the laboratory bench at 20-25°C for 9 d, one Penicillium expansum Link (ATCC 7861) isolate grown on the laboratory bench for 9 d and Rhizopus oryzae Went and Prinsen Geerligs grown on the laboratory bench for 3 d. The two isolates of M. jructicola were designated Mf#l (ATCC 32670) and Mf#2 which were originally isolated from infected cherries at the Summlerland Research Station. The spores were harvested asceptically with a cool loop and placed in 10 mL of sterile-distilled water (SDW). When enough spores were added to slightly color the SDW 50 mL drops replicated 6 times were placed on amended and nonamended PDA and allowed to incubate for 24 h at 20°C. Twenty-four hours later the number of germinated spores were counted out of 50 in each drop for a total of 300 spores. Spores were considered germinated if the germ-tube was at least as long as the spore. The data was analyzed using the SAS probit procedure (SAS Institute Inc., 1985) to determine the effective dose (ED) of hinokitiol required to prevent germination of 50% of the spore population.

Fungicidal Effect of Hinokitiol on M. fructicola Since M. jructicola is the major cause of postharvest decay in peaches this fungus was selected for the fungicidal test. The method used was adapted from Hoy and Ogawa (1984). Concentrations of H-D were made up from 0-20 J.tg/mL active ingredient of hinokitiol in SDW. Spores from Mf#l plates growing at 22°C for 9 d were harvested and a suspension of 3-5 x 104 spores/mL was obtained in each of 0, 10, 12, 14, 16, 18 and 20 j.tg/mL hinokitiol solutions. The solutions were incubated at 20°C for 20 h. After 274 / Sholberg and Shimizu

20 h they were centrifuged at 1188 x g for 90 s. The spore pellet was rinsed in SDW five times by discarding the supernatant, resuspending the pellet in 10 mL of SDW, and centrifuging for 90 s. Finally, 50 j.tL-drops of each suspension were pipetted onto PDA maintained at 22°C and the number of germinated spores were counted 24 h later. The above experiment was repeated with Mf#2.

Effect of Hinokitiol on Mycelial Growth Rate The effect of hinokitiol on the mycelial growth rate of M. jructicola and B. cinerea was tested by removing a 5 mm diameter disk from the margin of a 7-day-old culture and placing it in the center of a 9 cm diameter Petri plate containing 15 mL of PDA amended with 10-40 j.tg/mL active ingredient of hinokitiol. After incubation at 20°C for 7 and 3 d for M. jructicola and B. cinerea, respectively, on six replicate plates of each fungicide concentration, growth rate was determined as the average colony radius of two independent measurements divided by the number of days the the cultures had been growing. Regression analysis of each isolate was determined using the SAS regression procedure (SAS Institute Inc., 1985).

Effect of Hinokitiol on Peaches Contaminated with Decay Fungi Commercially mature redhaven peaches were obtained from five peach growers in the Okanagan Valley of British Columbia. Twelve peaches from each grower were treated with 300 j.tg/mL of hinokitiol dissolved in ethanol (H-L) or were placed upon a sheet of rayon paper treated with H-D for a concentration of 150 mg/m 2 (15 mg hinokitiol/m 2) and covered with cellophane paper coated with H-D at a concentration of 100 mg/m 2 (l0 mg hinokitiol/m2). Collectively these sheets of paper are termed "freshsheets" and were used to produce hinokitiol in the vapor phase. The peaches were incubated at 20°C for 10 d when the number of infected peaches were recorded. Peaches were considered infected with Monilinia spp. when buff colored colonies appeared on the fruit surface usually in concentric circles. Commercially mature redhaven peaches were randomly selected from the B.C. Fruit Packers' packinghouse at Summerland, BC. The peaches were decontaminated by dipping in a 10% (v/v) household bleach solution (5% NaOCl) for 2 min and rinsing for 2 min in cold tap water. Once the peaches had dried, samples were sprayed with a water suspension of 4 x 104 spores/mL Mf#2 in one experiment and with 5 x 104 Rhizopus spp. spores/mL in a similar experiment. Each peach was sprayed until it appeared wet which corresponded with approximately 1 mL of spore suspension. After the spore suspension had dried the peaches were treated by spraying 300 J.tg/mL Can. Ins!. Food Sei. Techl1ol. J. Vol. 24, No. 5, 1991

Table 1. Effective dose (ED) of hinokitiol (pg/mL) required to prevent germination of 500/0 of the spore population of three postharvest decay fungi. 95010 Fiducial Limits Upper Lower Fungus Dose (pg/mL) 25.3 37.7 30.0 Botrytis cinerea 13.5 15.8 14.7 Monilinia fructicola (Mf#l) 17.5 18.8 18.1 Rhizopus oryzae

H-L (approx. 1 mL per peach) or by placing between "freshsheets". Each treatment consisted of three replicates of 12 peaches. The experiment was repeated in September, 1990 with Mf#2 on more mature redhaven peaches. High concentrations of H-L, 500 and 1000 ~g/mL were tested as a dip in addition to freshsheets. The peaches were inoculated with 1 x 105 spores/mL of Mf#2 from 7-day old cultures growing on POA.

Results and Discussion Effect of Hinokitiol on Spore Germination Hinokitiol prevented M. fructicola, B. cinerea, R. oryzae and P. expansum spores from germinating. The M. fructicola isolate (Mf#l) was the most sensitive to hinokitiol with an E0 50 value of 14.7 ~g/mL (Table 1). Rhizopus oryzae with an E0 50 of 18.1 ~g/mL was almost as sensitive as M. fructicola. However Botrytis cinerea with an EO 50 of 30.0 ~g/mL was much less sensitive. The E0 50 of P. expansum was not determined since its spores were not sufficiently inhibited by hinokitiol concentrations below 40 ~g/mL. Hinokitiol acts on P. expansum over a relatively narrow concentration range between 35-40 ~g/mL since at 36 ~g/mL spore germination was reduced to 2.7 ± 1.9010 while at 40 ~g/mL, 100%

Table 2. Percent germination of Monilinia fructicola sporesl when suspended in hinokitiol. Percent germination

Hinokitiol (pg/mL)

Mf #1

o

97.0 6.7 61.0 0.3 11.7 72.7 0.0

10 12 14 16 18 20

± 1.5 2 ± 3.6 ± 7.6 ± 0.7 ± 4.8 ± 8.0 ± 0.0

Mf #2 20.7 2.4 2.0 2.4 1.6 1.0 0.0

± 4.2 ± 1.4 ± 1.6 ± 1.8 ± 0.8 ± 1.6 ± 0.0

ISpores were suspended in water suspensions of hinokitiol for 20 h at 20°C, washed five times with SDW and plated on PDA at 22°C for 24 h. 2Mean spore germination and standard deviation of six replicate samples.

inhibition was obtained. In order to determine the E050 of P. expansum spore germination tests at several concentrations between 35-40 ~g/mL would be required. Hinokitiol caused germ-tube distortion in B. cinerea spores. Hyphal enlargement and shortening of the cells adjacent to the spore occurred on POA containing 30 ~g/mL of hinokitiol after incubation at 22°C for 24 h (Figure 1). Germ-tube growth was severely reduced and confined to a 0.2 mm area on the hinokitiol amended medium, whereas on unamended POA the germ-tube was profusely branched and had grown well beyond 0.3 mm. This could indicate that hinokitiol caused abnormal cell division. The effect of hinokitiol on M. fructicola spores was fungicidal since exposure of spores to 20 ~g/mL hinokitiol and subsequent washing with SOW, resulted in none of the spores germinating (Table 2). This result indicates that the spores were killed by hinokitiol since its removal did not allow germination. Lower concentrations of hinokitiol were variable in its effect on M. fructicola spores. Hoy and Ogawa (1984) found similar variability with the surfactant, Nacconol on B. cinerea spores and were not able to calculate E050 values. It appears that hinokitiol is fungicidal at concentrations slightly higher than those at which it prevents spore germination.

Effect of Hinokitiol on Mycelia Growth Rate Rate of growth of M. fructicola isolates MF#I and MF#2 and B. cinerea on POA amended with hinokitiol decreased linearly as the concentration of hinokitiol was increased (Table 3). The following regression equations for M. fructicola (Mf#I, Eqn. 1 and Mf#2, Eqn. 2) and B. cinerea (Eqn. 3) describe the relationship between radial growth rate (mm/d) and concentration of hinokitiol ~g/mL), respectively: Fig. 1. Botrytis cinerea spore with germ-tube on PDA amended with 30 J.lg/mL hinokitiol after 24 h at 22°C. Note the hyphal enlargement and shortening of the cells in the germ-tube. Bar = 0.05 mm. Can. Insf. Food Sci. Techllol. J. Vol. 24, No. 5, 1991

y = -0.32X + 9.33

(I)

y = -0.32X + 11.05

(2)

+ 24.61

(3)

y = -0.31X

Sholberg and Shimizu / 275

Table 3. Mean radial growth rate and regression analysis of M. jructicola (Mf#1 and Mf#2) and B. cinerea on PDA amended with hinokitiol. Mean radial growth rate (mm/day)l Hinokitiol Mf #I Mf #2 B. cinerea (Jig/mL) 12.0 ± 0.3 25.0 ± lA 11.4 ± 0.02 o 5.7 ± 0.0 7.8 ± 004 10 5.6 ± 0.1 704 ± 0.1 12 4.6 ± 0.5 7.1 ± 0.1 14 17.6 ± lA 4.3 ± 0.6 6.5 ± 0.5 16 2.3 ± 004 4.1 ± 0.6 18 1.3 ± 0.3 0.0 ± 0.0 20 18.2 ± 1.2 24 17.1 ± 0.2 28 14.7 ± lA 32 12.8 ± 3.7 36 0.0 ± 0.0 0.0 ± 0.0 40 Regression analysis of growth rate vs. concentration R2 0.74 0.86

0.70

Parameter estimates Intercept Standard error Significance

9.3261 004205 0.0001

11.0525 0.2799 0.0001

24.6049 0.9289 0.0001

-0.3213 -0.3208 -0.3055 Slope Standard error 0.0237 0.0157 0.357 Significance 0.0001 0.0001 0.0001 1M. jructicola growth rate was calculated after 7 days and B. cinerea after 3 days. 2Mean radial growth rate and standard deviation of six replicates.

The calculated E050 value for mycelial growth rate or the growth rate at which the mycelium was inhibited by 50010 was 14.6, 17.3 and 39.7 Itg/mL hinokitiol, for M. jructico/a isolates Mf#l and Mf#2 and B. cinerea respectively. The E050 value for M. jructico/a (Mf#l) at 14.6 Itg/mL was slightly less than for spore germination at 14.7 Itg/mL indicating that its mycelium was more sensitive and would be controlled by a slightly lower concentration. Mycelial growth of B. cinerea required relatively high concentrations of hinokitiol to be affected showing again that it was less sensitive to hinokitiol than M. jructico/a. Although the E050 values of R. oryzae or P. expansum were not calculated their mycelial growth rates were inhibited at 36 Itg/mL hinokitiol.

Control of Decay on Harvested Peaches Peaches from a grower's orchard naturally contaminated with M. jructico/a spores did not develop brown rot or any other postharvest disease when they were covered with hinokitiol freshsheets. In comparison, 41.1 % of the control fruit from the same orchard did develop brown rot. This result indicates peaches from commercial fruit growers can be protected from decay causing organisms with hinokitiol. Hinokitiol was effective in reducing brown rot and Rhizopus rot on peaches that had been inoculated with M. jructico/a or Rhizopus spp. spores in 1989 (Table 4). Hinokitiol freshsheets were more effective than hinokitiol dissolved in ethanol in preventing Rhizopus rot and brown rot. In a further experiment (i.e. 1990), hinokitiol dissolved in ethanol was ineffective in controlling brown rot on over-mature peaches but hinokitiol-containing freshsheets reduced brown rot by 44.3%. Perhaps hinokitiol, in the gaseous state, is more toxic to fungal spores than when it is dissolved in ethanol or water. More research is required on the volatility of hinokitiol, and its effectiveness when in this state. Hinokitiol has been used to preserve chinese mustard, lettuce, champignon mushroom, oyster mushroom and cut carnation flowers by wrapping the produce in freshsheets (B. N. Shimizu, unpublished data). Lemons soaked in a 0.1 % hinokitiol aqueous solution showed considerably less decay than lemons which were not treated (B. N. Shimizu, unpublished data). Hinokitiol offers promise as a new postharvest decay control chemical. Further development of this interesting natural product should be given high priority because of the need for postharvest disease control.

Acknowledgements We wish to. thank Eliza Yue for excellent technical assistance and Hitomi Wakabayashi for assistance in organizing some of the field experiments. We also thank Or. Tom Beveridge and Or. O. L. Lau for helpful suggestions.

Table 4. Percent brown rot and Rhizopus rot of harvested redhaven peaches treated with hinokitiol. 1990 1989 Treatment Concentration (Jig/mL) Brown rot Brown rot Rhizopus rot Control loo.0a n.2a 1 47.2a Hinokitiol in ethanol 300 58.3a 13.9ab Hinokitiol in ethanol 1000 100.0a Hinokitiol 13 .9b 11.1 b 56.7b freshsheet 2 IMeans followed by the same letter in each column are not significantly different using Duncan's multiple range test (P> 0.05). 2Freshsheets are sheets of paper treated with hinokitiol. In this experiment a sheet of rayon paper containing 150 mg/m 2 hinokitiol was placed under the peaches and cellophane paper coated with 100 mg/m2 hinokitiol was placed over the peaches. 276 / Sholberg and Shimizu

Can. Insl. Food Sci. Technol. J. Vol. 24, No. 5, 1991

References Avissar, I., Marinansky, R. and Pesis, E. 1989. Postharvest decay control of grape by acetaldehyde vapors. Acta Hortic. 258:655. Eckert, J .W., 1988. Historical development of fungicide resistance in plant pathogens. In: Fungicide Resistance in North America. C.J. Delp (Ed.). p. 1. APS Press, St. Paul, MN. Hoy, M.W. and Ogawa, J.M. 1984. Toxicity of the surfactant Nacconol to four decay-causing fungi of fresh-market tomatoes. Plant Dis. 68:699. Jones, A.L. and Sutton, T.B. 1984. Diseases of Tree Fruits. Cooperative Extension Service, Michigan State University, East Lansing, MI. Meheriuk, M. and McPhee, W.J. 1984. Postharvest handling of pome fruits, soft fruits, and grapes. Agriculture Canada, Publication 1768E, Ottawa, ant.

Call. hiS!. Food Sci. Technol. J. Vol. 24, No. 5, 1991

SAS Institute Inc. 1985. SAS User's Guide: Statistics, 1985 Edition. SAS Institute Inc., Cary, NC. Wilson, E.E. and Ogawa, J.M. 1979. Fungal, Bacterial, and Certain Nonparasitic Diseases of Fruit and Nut Crops in California. University of California, Berkeley, CA. Wilson, C.L., Franklin, J.D. and Otto, B.E. 1987. Fruit volatiles inhibitory to Monitiniafructicola and Botrytis cinerea. Plant Dis. 71 :316. Wilson, C.L. and Wisniewski, M.E. 1989. Biological control of postharvest diseases of fruits and vegetables: an emerging technology. Annu. Rev. PhytopathoI. 27:425.

Submitted January 28, 1991 Revised April 24, 1991 Accepted June 10, 1991

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