ISR 2000®: an aid to biocontrol of postharvest decay in citrus

Postharvest disease control in citrus is traditionally achieved by chemical means (Knight et al., 1997).

While a number of chemicals have in the past been used, the available options have in recent years become restricted. In some cases, resistance to fungicides, such as that of the benzimidizole group (Brown, 1977) by the most common citrus postharvest pathogen, Penicillium digitatum has occurred. This has left a very restrictive list of fungicides, with the most widely used being imazalil sulphate.

There is a potential for resistance to develop against this compound, and in fact there have recently been such reports, although unconfirmed. A further problem relating to the traditionally used postharvest fungicides is that of consumer resistance. This was noted some time ago by Droby et al. (1991), and has steadily become more important. Increasingly, food safety is considered an issue, and de-registration of some fungicides has recently taken place (Lopez-Garcia et al., 2000). This is likely to intensify. Already, juicing plants refuse fruit that has been treated with postharvest fungicides, and some markets pay a premium for non-treated fruit.

In order to address the problems of resistance, both fungal and consumer, alternative approaches to postharvest decay management have for some time been sought (Wilson, 1997). The likelihood of a new generation of chemicals becoming available is limited by development cost and consumer resistance. Therefore, for a number of years, biological approaches have been tried; and Wisniewski and Wilson (1992) have published a mini review on the subject. The most important biological approach has been the work with applications of various yeasts. Some products have even been registered for the purpose (Wilson, 1997).

However, success has been limited. The mode of action is considered to be one of competitive inhibition (Arras et al., 1998). However, most infections by wound pathogens take place between the orchard and packing, allowing at least a number of hours for the fungus to establish before the yeast is applied. As a result, effectiveness of the yeast is poor (Chalutz and Wilson, 1990).

This is sometimes countered by applying low dosages of fungicide (Chalutz and Wilson, 1990) to decrease growth potential of the fungus and allow establishment of the yeast in the wound site. This is not a long-term solution, as the possibility of resistance of the fungus to the fungicide is enhanced, and consumer resistance will remain. Citrus rind is known to have anti-fungal properties, which change with increasing maturity (Angioni et al., 1998). Pathogens such as P. digitatum usually do not grow on immature fruit. The ability to grow, however, changes rapidly after color break, and fruit becomes increasingly susceptible with age.

Differences in ability to withstand fungal attack are also evident between orchards, as well as seasons.

If the anti-fungal characteristics of citrus fruit can be enhanced, the likelihood of postharvest growth of the pathogenic fungi causing decay could be decreased. If it is not possible to decrease decay to economic levels without any other intervention, the additional use of a postharvest yeast application may be more successful, as the ability of the yeast to establish and thus compete with the fungus may be increased.

It is known that anti-fungal compounds can be developed in fruit postharvest by means of ultraviolet irradiation (Droby et al., 1993). However, this may be too late, and thus such a system has not been a commercial success. Nevertheless, the possibility of enhancing the anti-fungal properties of the rind preharvest would solve the problem and make the possibility of an integrated postharvest disease control technique without the use of chemical fungicides a possibility.

Wild (1993) found a significant increase in resistance of Valencia oranges to P. digitatum after dipping fruit in a solution of potassium phosphonate, and believed that this was due to an enhancement of anti-fungal substances in the rind. The product ISR 2000® is thought to be a phytoalexin enhancer, and at the same time is acceptable for the production of organic produce, making it very attractive for use where market sensitivity to chemical fungicides may exist.

The intention of the work being reported was to evaluate the efficacy of ISR 2000® as an aid to replacing other postharvest disease control compounds, or as an aid to enhancing postharvest biocontrol.


Materials and methods

The fruit used in the work was obtained from a commercial orchard growing navel oranges (Citrus sinensis) situated near Pietermaritzburg, South Africa. The trees were in good condition. The work was done late in the season, when fruit could be expected to have an enhanced susceptibility to postharvest fungal diseases.

For preharvest applications of ISR 2000® with the intention of enhancing endogenous resistance of fruit to postharvest decay, a randomised block design was used, with five replications and two trees per plot. Preharvest treatments were 1) control: Trees were sprayed to runoff with water. These trees were sprayed before any other applications were made, but using the same equipment, and 2) ISR 2000®: applied after mixing the product at 50 mls per 100 L water. Trees were again sprayed to runoff. Samples were picked for further treatment 14 and 21 days after the preharvest applications.

Postharvest treatments consisted of 1) Control: Fruit were dipped in distilled water for one minute, 2) Imazalil: Fruit was dipped for one minute in a solution of imazalil sulphate at a concentration of 500 ppm (This treatment served as the commercial control.), 3) and 4) Yeast: A commercially prepared dried product containing Cryptococcus albidus originally isolated from the fruit surface of stone fruit was used. Two concentrations, consisting of 2 and 4 g dry product per L water were used. The dry product was rehydrated in water at 36°C and allowed to stand for 20 minutes before use. A wetter was added to aid application.

For each treatment, five replications consisting of two fruits per replication were used. In each case, fruit were inoculated with P. digitatum at 106 spores per ml. Inoculations were made using a sharp object dipped into the spore suspension, which penetrated the rind including both flavedo and albedo. The number of spores introduced into the wound were deemed sufficient to ensure infection, which was checked in the control fruit. Four inoculations per fruit were made.

After fruit were inoculated, they were left for 3 hrs before further treatment as outlined, so as to simulate the time elapsing between harvest and arrival at a packing facility. Once treated, the fruits were allowed to dry before being placed in paper packets, and left at 20°C for 10 days, after which they were evaluated for fungal growth. The extent of fungal growth was scored on a scale of 1 to 5, where 1 indicated no visible growth of fungal mycelium, and 5 coverage of the entire fruit surface.


Results

For both the 14-day and 21-day post-treatment sampling dates, control fruit showed a high degree of infection, demonstrating that resistance of the fruit to inoculated P. digitatum was low (Tables 1 and 2). Imazalil, as an industry standard for postharvest disease control, was highly effective.

The yeast application postharvest appeared to have some effect, but was variable. From the first sampling date (Table 1), it appeared that the lower concentration of yeast was not very effective if no preharvest treatment of ISR 2000® was applied. The higher concentration, however, was somewhat better, having significantly (P=0.01) lower waste than the control, although probably not good enough as a commercial control. Similar results were noted at the 21-day sampling date (Table 2), but with no improvement in effectiveness with higher concentration.

The effect of ISR 2000® was marked. For fruit not treated postharvest, the second sampling (21 days) was clearly superior. This may indicate a longer period is necessary to elicit the desired plant reaction, with direct effect on the pathogen. It is concluded that an induced response in the fruit had occurred. In combination with yeast, postharvest disease control was as effective as that of the imazalil-treated fruit, with no statistical difference between this and the ISR 2000® plus yeast combination.


Table 1. Effect of pre- and postharvest treatments on growth of Penicillium digitatum 14 days after treatment with ISR 2000®.


¹Infection score: 1 indicates no growth and 5 indicates complete coverage of the fruit by mycelium.
^a,b,c,d,e means differ LSD (P=0.05) = 0.54 and (P = 0.01) =0.71.



Table 2. Effect of pre and postharvest treatments on growth of Penicillium digitata 21 days after treatment with ISR 2000®.


¹Infection score: 1 indicates no growth and 5 indicates complete coverage of the fruit by mycelium.
^a,b,c,d means differ LSD (P = 0.05) = 1.13 and (P = 0.01) = 1.48.



Taking into account the harshness of the inoculation procedure, this combination was effective, and could be considered commercially acceptable. The higher concentration of yeast in combination with ISR 2000® would probably be advisable to ensure adequate competition with the fungus. While postharvest applications of yeast may not always be effective in controlling decay in citrus, in combination with ISR 2000®, there is a real possibility that commercial control can be effected without the use of traditional fungicides.

References

Angioni, A., P. Cabras, G. D’Hallewin, F.M. Pirisi, F. Reniero and M. Schirra. 1998. Synthesis and inhibitory activity of 7-geranoxycoumarin against Penicillium species in citrus fruit. Phytochemistry 47:1521-1525.

Arras, G., V. De Cicco, S. Arru and G. Lima. 1998. Biocontrol by yeasts of blue mould of citrus fruits and the mode of action of an isolate of Pichia guilliermondii. J. Hort. Sci. and Biotechnology 73:413-418.

Brown, G. 1977. Application of benzimidazole fungicides for citrus decay control. Pro. Int. Soc. Citriculture. 1:273-277.

Chalutz, E. and C.L. Wilson. 1990. Postharvest biocontrol of green and blue mould and sour rot of citrus fruit by Debaryomyces hanmsenii. Plant Dis. 74:134.

Droby, S., E. Chalutz and C.L. Wilson. 1991. Antagonistic microorganisms as biological control agents of postharvest diseases of fruits and vegetables. Postharvest News and Information 2:169-173.

Droby, S., E. Chalutz, B. Horev, L. Cohen, V. Gaba, C.L. Wilson and M. Wisniewski. 1993. Factors affecting UV-induced resistance in grapefruit against the green mould decay caused by Penicillium digitatum. Plant Path. 42:418-424.

Knight, S.C., V.M. Anthony, A.M. Brady, A.J. Greenland, Sp.P. Heaney, D.C. Murray, K.A. Powell, M.A. Schulz, C.A. Spinks, P.A. Worthington and D. Youle. 1997. Rationale and perspectives in the development of fungicides. Ann. Rev. Phytopathol. 35:349-372.

Lopez-Garcia, B., L. Gonzalez, E. Perez-Paya and J.F. Marcos. 2000. Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits. The American Phytopathological Soc. 13:837-846.

Wild, B. 1993. Cyclohexamide and phosphonate effects on the susceptibility of citrus fruit to green mold decay. Acta Hort. 343:353-356.

Wilson, C. 1997. Postmodern fungicides – breaking out of the synthetic fungicide paradigm. Proc. Australasian Postharvest Horticulture Conference Sept. 1997, pp. 132-138.

Wisniewski, M.E. and C.L. Wilson. 1992. Biological control of postharvest diseases of fruits and vegetables: Recent advances. Hort. Science 27:94-98.