Effectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots

Postharvest Biology and Technology ELSEVIER

Postharvest

Biology

IO ( 1997) 169- 178

and Technology

Effectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots Giuseppe Lhpurtrmento

Lima’,

Antonio

Ippolito,

dt Protr~~one deile Ptante dalle Malattte.

Recewed

3 April

UnxwsitZl

Franc0

Nigro,

degli Stud1 t/r Barr.

1996: accepted

16 September

Mario

Lra Ammdola.

Salerno* 165, ‘3. 701.X Bar/. Ittrlj

1996

Abstract Many yeasts, including yeast-like fungi, were selectively isolated from fruits and vegetables. In several assays performed on strawberries, table grape berries and kiwifruit, the yeast-like fungus Aureobasidiunz pulllh~~ L47 and the yeasts Candida vanderwaltii L60 and C. oleophila L66 were the most effective antagonists of Botrytis cinerra and Rhizopus stolonifer. Isolates L47 and L66 were utilized in trials on strawberries grown under plastic tunnels. They were applied at flowering (full bloom and late petal fall) and at fruit maturity (just before or after harvest). Isolate L47 was the most effective against both B. cinerea and R. stolonifer. Both antagonists were more active when applied at the flowering stage. with isolate L47 more effective than vinclozolin. The antagonist population was consistently high on flowers and developing fruits, and on cold-stored strawberries. Isolates L47 and L66 showed a low sensitivity towards some fungicides in culture and were able to grow at temperatures between those of cold storage and 33°C. Competition for nutrients seems to be the main mode of action. ~(3 1997 Elsevier Science B V. Kqwvrd.s: Aurrobasidzunz pullulans; Biological control; Botrytrs Rhimpus .stoloniftr: Strawberry; Yeasts; Yeast-like fungi

1. Introduction Fungicide method

for

treatments controlling

have

been

postharvest

the

97?$17.00


PIZ SO9’1-5214(96)01302-6

Science

B.V. All rights

fruits

and

vegetables.

about

residues,

and

oleophila:

Postharvest

However, the progressive

public loss

diseases:

concern of effec-

due to selection of fungicide-resistant isolates of pathogens, have increased the search for alternative means less harmful to human health and to the environment. In the postharvest environment, more than in any other stage of production, biological control by means of micro-organisms naturally occurring on fruits and vegetables is a very promising alter-

of

* Corresponding author. Fax: + 39 80 5442813: e-mad [email protected] I Present address: Dipartlmento di Scienze Animali Vegetali e dell’Amblente. Umversitl degh Studi de1 Molise, viale Manzom. 86100 Campobasso, Italy. 0925.5213

Candidu

tiveness

main

diseases

ckerru:

reserved

170

G. Lima et al. / Postharvest

Biology and Technology

native to fungicide application (Wisniewski and Wilson, 1992). At present, yeasts appear the most promising biocontrol agents because competition for nutrients, direct interaction with the pathogen and induction of host defence seem to be the main modes of action (Droby et al., 1994); these mechanisms make yeast biocontrol agents readily acceptable by consumers, since production of antibiotics seems not to be involved. Moreover, yeast activity may be enhanced by integration with cultural methods (Sugar et al., 1994), low fungicide concentrations (Droby et al., 1993) or calcium salts (Ippolito et al., 1994). The yeast-like fungus Aureobasidium pullulans (de Bary), Arnaud seems a possible biocontrol agent because of its ability to colonise a wide range of hosts (Webb and Mundt, 1978) and to survive under various environmental conditions (Kohl and Fokkema, 1994). The aims of this study were to evaluate under field conditions the effectiveness of two antagonists (A. pullulans L47 and Candida oleophila Montrocher L66) against postharvest strawberry rots due to Botrytis cinerea Pers: Fr. and Rhizopus stolonifer Ehrenb, and to find a suitable control strategy. The dynamics of the antagonist population were monitored and observations were made on modes of action, growth temperatures, and sensitivity towards some fungicides.

2. Materials and methods 2.1. Isolation and screening of antagonists

Selective isolation of yeasts and yeast-like fungal antagonists of B. cinerea was performed on table grape, strawberry and kiwifruit as described by Wilson et al. (1993). The most effective antagonists were further tested on table grape berries (cv. Italia) wounded by removing the pedicel, and on strawberry fruits (cvs. Chandler and Pajaro) and kiwifruits (cv. Hayward) injured in the equatorial zone with one and two wounds (3 mm wide x 3 mm deep), respectively. A water suspension (30 ~1) of antagonist cells (5 x lo’- lo* cfu/ml) was placed in each wound and, after 2 h at room temperature, the fruits

10 (1997) 169-178

were inoculated separately by placing 15 ~1 of B. cinerea, or R. stolonifer or Aspergillus niger van Tiegh. spore suspensions (1.5 x lo4 spores/ ml) in each wound. Fruits were maintained at 20°C at high RH and decay was evaluated after 5 days. Each treatment consisted of five replications and each replicate consisted of ten table grape berries, six strawberry fruits or six kiwifruits. The assays were performed at least twice. 2.2. Field trials The trials were conducted on the strawberry cultivars Chandler and Pajaro grown under plastic tunnels in two commercial strawberry groves located in Southern Italy at Policoro (Province of Matera) and at Conversano (Province of Bari) respectively. Two of the most effective antagonists, A. pullulans L47 and C. oleophila L66 (5 x lo’- lo8 cfu ml - ‘), were sprayed on labelled flowers at full bloom (Conversano, 10 April; Policoro, 4 May), and at late petal fall (Conversano, 13 April; Policoro, 8 May), or on strawberry fruits just before or just after harvest (75% full red colour). In the later trial the antagonist was sprayed directly on both sides of the fruits arranged in a single layer in plastic trays. In addition to the untreated control in the flower sprays, a treatment with Ronilan (vinclozolin 50% a.i.) at 100 g/100 1 was also included. For each treatment, individual plots were 0.5 m wide and 15 m long mulched rows, spaced 1 m apart arranged in a randomised complete block design with four replications. On each plot, 150 strawberries at a maturity stage suitable for cold storage were harvested, placed in plastic trays (five fruits per tray) in a single layer and stored for 7 days in a cold room at 3 f 1°C and 95-98% RH. After a shelf-life of 5 days at 20°C the percentage of decayed fruits due to B. cinerea or R. stolonifer infection were recorded. On fruit from the treatment at flowering, B. cinerea infection originating from the zone of senescing floral parts and that originating from surface lesions on fruits were recorded separately. During the trials, daily temperature, RH and rainfall were recorded.

G Lma et al. 1 Postharvest Blolog_r and Techttologv 10 (I 997) 169- 178

2.3. Microbial epiphvtic population The microbial epiphytic population was evaluated on strawberry flowers and fruits from the field at Policoro. In particular, the first samples to estimate the population were taken 2 h after the application of the antagonists (4 May), while the second samples were taken immediately before the second application (8 May). For each plot, four samples of ten flowers or ten fruits gathered at random were processed. Each sample suspended in 200 ml of sterile distilled water was placed on a rotary shaker at 200 rpm for 30 min. A sample of washing water (0.1 ml) was diluted and poured on basal yeast agar (BYA) plates (Fokkema et al.. 1987) containing 250 mg/l of ampicillin and 250 mg/l of streptomycin sulphate to isolate yeasts and filamentous fungi, and on nutrient yeast-extract dextrose agar (NYDA) supplemented with 200 mg/l of cycloheximide to isolate bacteria. The plates were incubated at 25°C and after 3-4 days, the colonies of yeast, yeast-like fungi, filamentous fungi and bacteria were recorded separately, although the yeasts and the yeast-like fungi are reported and discussed together. 17.4. Characterisation of the antagonists. The possible mode of action of C. oleophila L66 and A. pullulans L47 was investigated in vitro and in vivo. The production of antibiotics was evaluated both in Petri dishes and on injured table grape berries (cv. Italia). In the former case, the antagonist was grown on NYDA and on potato dextrose agar (PDA) in the presence of B. cinereu and the size (mm) of any inhibition zone was recorded after 5-7 days at 20°C. An isolate of Bucillus subtilis (Bs51) was included as a control. In vivo activity of the antagonist culture filtrates against B. cinereu, as well as that of the killed (autoclaved) cells, was evaluated in comparison with the activity of the viable antagonist cell. The culture filtrate was obtained by growing the antagonist in NYD-broth (NYDB) for 48 h, then discarding the cells from the medium by centrifugation (5000 x g for 15 min) and filtration through a 0.2 pm sterile filter. The in vivo observations were performed on table grape berries

171

injured by removing the pedicel, using the technique previously described for antagonist screening on wounded fruit. To investigate the competition for nutrients, germination of B. cinerea conidia was evaluated on table grape peel leachates at different nutrient concentrations and in the presence of the antagonist cells, as described by Droby et al. (1989). The peel leachate was obtained by shaking, in 100 ml of sterile distilled water, ten table grape berries whose peel was pricked with a needle (five wounds per berry). Moreover, on the table grape berries injured by removing the pedicel, treated with the yeasts and inoculated with B. cinereu, the effect of exogenous nutrients (20 ~11 of NYDB introduced in each lesion just after pathogen inoculation) on antagonist activity was also evaluated. The growth of the antagonist was evaluated at 0, 4, 10, 15, 25, 30, 33, 35 and 37°C. For each antagonist, 100 ~11 of cell suspension, containing about 100 cells, were poured on NYDA Petri dishes; the plates (four replicates per temperature) were incubated for 10 days and the antagonist colonies were counted. The sensitivity to some fungicides was evaluated on BYA medium. Each fungicide, suspended in distilled water, was mixed in Petri dishes with the medium at 45°C to obtain the final concentrations of 0, 10, 100 and 500 mg:‘l of commercial formulates. The plates (four replications per treatment) were covered with 100 ,~l of the antagonist suspension containing about 100 cells. The antagonist colonies were counted after 7 days of incubation at 24°C. -7.5. Statistical analysis The results were submitted to variance analysis and the mean values were compared using Duncan’s multiple range test. Percentages were converted into Bliss angular values (arcsin V,‘“%) before analysis.

3. Results More than 200 yeasts and yeast-like fungi were selectively isolated from different fruits and veg-

172

G. Lima et al. / Postharvest

Biology and Technology

etables. In the assays on wounded strawberries, table grape berries and kiwifruits, isolates L47 and L60 obtained from table grape, and L66 from strawberry, identified as Aureobasidium pullulans (de Bary) Arnaud, Candida vanderwaltii (VidalLeira) Meyer and Yarrow and C. oleophila Montrocher., respectively, were observed to be the most effective antagonists of B. cinerea. Moreover, these isolates also showed an appreciable activity against R. stolonifer and A. niger. Isolates L60 and L66 were identified by the Centraalbureau Voor Schimmelcultures (CBS, Delft, The Netherlands) and deposited as collection numbers 8270 and 8269, respectively. Isolate L47 of A. pullulans was also deposited at the CBS-Baarn (collection number, 351.96). Botrytis and Rhizopus rot of strawberry fruits from plants treated with isolates L47 and L66 at the flower stage are shown in Fig. 1. Botrytis rot data from Policoro only are plotted since the results were very similar on both experimental fields. Rhizopus rot data were collected only at Policoro because of the low incidence of the pathogen on strawberries harvested about 20 days earlier in the other experimental field at Conversano. Both antagonists applied at the flower stage significantly reduced Botrytis rot originating from senescing floral parts. Isolate L47 was more effective and reduced infection by 84% in the untreated control. It showed significantly higher activity than vinclozolin. Only isolate L47 significantly reduced (by 26%) Botrytis rot originating from the surface lesions on the fruit. Isolate L47 was also effective against rot due to R. stolonifer and reduced infection by 78%. Isolate L66 and vinclozolin were both ineffective against Rhizopus rot. On fruits treated immediately before harvest, isolate L47 reduced Rhizopus rot by 72% compared with the control while only a slight activity was observed against Botrytis rot. Isolate L66 reduced Rhizopus rot but not significantly (Fig. 2). On fruits treated immediately after harvest but while still in the field, isolates L47 and L66 reduced Botrytis rot by 40 and 19%, respectively, but only isolate L47 significantly reduced Rhizopus rot (Fig. 3).

10 (1997) 169-l 78

Total epiphytic yeast (including yeast-like fungi) populations (1 06- 1O7 cfu/fruit on strawberry fruit treated at both the flower and harvest stages with either L47 or L66) were consistently high except at the 8 May assessment. The population of total filamentous fungi (principally Cladosporium, Botrytis, Rhizopus and Penicillium, spp. ) was reduced by isolate L47 on strawberries treated at the flower stage (Figs. 1 and 3). The epiphytic bacterial population was highly variable (data not shown). Isolate L47 induced small dark superficial lesions on a few strawberry fruits and then only among those harvested after the correct maturity stage. Moreover, isolate L47 induced a high suppression of B. cinerea sporulation both on strawberries and on table grapes and kiwifruits. The size of the inhibition zone exerted by C. oleophila L66, A. pullulans L47, and B. subtilis Bs51 was on average 0, 1 and 8 mm, respectively. Isolates L47 and L66 showed no antagonistic activity against B. cinerea when assayed as killed cells on wounded table grape berries (Table 1). Exogenous nutrient (NYDB) introduced in the wounds lowered the activity of the viable antagonist cells. The culture filtrate of isolate L66 did not influence the development of Botrytis infection, while that of isolate L47 showed only a slight, non-significant activity against the disease (Table 1). The viable cells of both antagonists significantly reduced the germination percentage of the B. cinerea conidia when suspended in table grape peel leachate, in conditions of low nutrient concentrations; conversely, in the presence of high nutrient concentrations, obtained by adding grape juice to the medium, the germination of B. cinerea conidia was not affected (Table 2). Microscopic observations revealed that when nutrient concentration was low, the cells of C. oleophila L66 adhered to B. cinerea mycelium and germinating conidia. Isolate L47 grew in culture at temperatures from 0 up to 33°C isolate L66 from 4 to 33°C. A temperature of 35 or 37°C was lethal for both antagonists as shown by absence of growth when

G. Lima

et al.

! Postharvest

Biology

and Technolog~~ IO (1997)

169- 178

L66

-A-

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^

+

1

A

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Fungicide

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Untreated

-

Temp.

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1

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Fig. 1. Botrvtis and Rhizopus rots (A, B) and flower stage (full bloom, 4 May and petal fall, assessment was made on fruits harvested on 30 factors are also reported (E). Mean separation capital letters.

populations of yeasts and filamentous fungi (C, D) on strawberry fruits treated at 8 May ) with A. pullulans L47, c‘. olrophila L66 and fungicide (vinclozolin). Disease May, stored for 7 days at 3°C followed by 5 days of shelf-life at 20°C. Some climatic according to Duncan’s multiple range test at P I 0.05, small letters, and P 5 0.01,

plates were subsequently incubated at room temperature. The minimum inhibitory concentration (mg/l) of some fungicides towards the in vitro growth

of A. pullulms L47 and C. oleophih L66 is shown in Table 3. Both L47 and L66 were most sensitive to the MancozebjRidomil mixture.

G. Lima et al. /Postharvest Biology and Technology 10 (1997) 169-178

174

g

10 ,,,_,_,.,.,.,_,., ,.,,,_,.,.,_(_,.,.‘.‘.‘,..‘,.,‘.‘.. .‘.‘.‘,‘,‘.‘,‘,‘,’

0 L47

L66

Untreated

Fig. 2. Botrytis and Rhizopus rots on strawberry fruits treated with A. pullulans L47 and C. oleophila L66 immediately before harvest. Disease assessment was made on fruits stored for 7 days at 3°C followed by 5 days of shelf-life at 20°C. Mean separation according to Duncan’s multiple range test (P 2 0.05).

4. Discussion More than 200 isolates of yeasts and yeast-like fungi with activity against B. cinerea were obtained from fruits and vegetables in Southern Italy. Isolates L47, L60 and L66, identified as A. pullulans, C. vanderwaltii and C. oleophila, respectively, were the most effective against B. cinerea and R. stolonifer in assays on wounded fruit. The yeast-like fungus A. pullulans is one of the most widespread and well-adapted saprophytes in the phyllosphere (Blakeman and Fokkema, 1982) and was isolated from fruit washings. It is one of the most frequent micro-organisms on fruits such as grapes (Davenport, 1976), strawberries (Buhagiar and Barnett, 1971) and on various vegetables (Webb and Mundt, 1978). A. pullulans is an effective nutrient competitor in the phyllosphere (Fokkema, 1973) and has been considered as a possible biological control agent against a range of pathogens, including B. cinerea on strawberry (Bhatt and Vaughan, 1962). The yeast C. oleophila, isolated from fruits, proved to be effective against B. cinerea on wounded apples (Mercier and Wilson, 1995), and it is now patented as a biological control agent against some postharvest diseases of fruits and vegetables (Wilson and Wisniewski, 1994). The yeast C. vanderwaltii has not previously been reported as a biological control agent. A. pullulans L47 and C. oleophila L66 showed more activity against B. cinerea rots when applied at bloom in comparison with application at har-

vest. Our results have confirmed that most Botrytis rots on strawberries start on infected, senescent floral parts leading to the latent infections that later develop into active rots on ripe fruits (Powelson, 1960). Isolates L47 and L66 were shown to colonise the flower to the extent that their antagonistic activity prevented colonisation of senescent floral parts by B. cinerea. The pathogen was frequently isolated in culture from the senescent stamens of untreated fruits, but only seldom from fruit treated with the antagonists or with vinclozolin. B. cinerea rots originating from surface lesions on the fruit epidermis were adversely affected only by isolate L47, which had the highest population on the fruits. When applied to strawberries just after harvest, both isolates L47 and L66 reduced Botrytis infections, but when applied to fruits still on the plant, and harvested just after drying, only isolate L47 was observed to have any effect. The lesser uniformity of treatment on fruits still on the plant may account for the lower antagonistic activity observed in the field and better results could perhaps be obtained with other strawberry growth systems or different application technology. Isolate L47 was effective against R. stolonifer also with applications at harvest. This difference from B. cinera can be explained on the basis of the different disease cycle of the pathogens. Unlike B. cinerea, R. stolonifer is seldom present on the flowers and infects only ripe strawberries (Harris and Dennis, 1980).

G. Lima et al.

I Postharvrst Biology and Technology IO (1997) 169-l 78

Botfytis rot

_

175

Rhizopub rot

:::: .,.,.,.,.,.,.,.,.,

,.,.,,,,,.,.,.,,,

L66

8

’

Untreated

’

L47

‘.‘.‘.’

.‘.‘,’ ‘.’ ‘.‘..a .‘,‘.‘,‘(‘.‘.‘.’

Untreated

Yeasts and yeast-like fungi

Fig. 3. Bofrytis and Rhimpus rots and populations of yeasts and filamentous fungr on strawberry fruits treated with il. pullu1un.s L47, C. oleophikr L66 immediately after harvesting. Disease assessment was made on fruits stored for 7 days at 3°C followed by 5 days of shelf-life at 20°C. The epiphytic microbial population was assessed immediately after the treatment (A), at the end of cold storage (B) and at the end of shelf-life (C). Mean separation according to Duncan’s multiple range test at Pi 0.05. small letters, and at P s.O.01, capital letters.

The data on the epiphytic microbial population of strawberry flowers and fruits and Petri plate cultures indicate that both isolates, particularly L47, have a good ability to survive under field (plastic tunnels) and storage conditions. The dip in population of both yeasts and filamentous fungi on 8 May, after a period of low RH started around 5 May, emphasises the importance of moisture in establishment of the antagonist and its biocontrol performance (Mercier and Wilson, 1995). Competition for nutrients, as a possible mode of action. seems to play an important role in the activity of isolates L47 and L66. This mechanism is an important feature for control of fungi such as B. cinerea, since the pathogen needs high levels of exogenous nutrients to germinate and to infect the host tissue (Blakeman and Brodie, 1977). In the

case of isolate L66, microscopic observation showed that a direct interaction with the pathogen could also be involved. Isolate L47 showed little antibiotic activity as demonstrated by both the lack of in vivo activity of the culture filtrate and the slight in vitro inhibition of the pathogen as compared with B. subtilis. According to Fokkema (1973) the in vitro inhibition zone produced by .4. pulluluns could also result from nutrient depletion in the zone of interaction with the pathogen. Production of antibiotics by isolates of A. pulfu/un.s has been reported (McCormack et al., 1994) but their role in antagonistic activity is not clear. In addition, as mechanisms of action for A. pulluluns, competition for space (Dickinson and Skidmore, 1976) and induction of host resistance (Flood and Rees, 1986) could be involved.

176

G. Lima et al. / Postharvest

Biology and Technology

10 (1997) 169-l 78

Table 1 Influence of treatment with A. pullulans L47 and with C. oleophila L66 on infection by B. cinerea of wounded, inoculated table grape berries

Table 3 Minimum inhibitory concentration (mg/l) of some fungicides towards in vitro growth of A. pullulans L47 and C. oleophrla L66

Treatments

Fungicide

L47

L66

Iprodione (Rovral, 50) Procymidone (Sumisclex, 50) Vinclozolin (Ronilan, 50) Copper oxychloride (Azuram, 40) Diclofluanide (Euparen, 50) Fenarimol (Rubigan 6PB, 6) Penconazole (Topas IOEC, 10) Mancozeb + Metalaxyl (Ridomil MZ, 6+64)

>100<500 > 500 > 500 > 500 >lO 500 >lO
>500 >500 > 500 > 500 1500 > 500 > 500 < 10

The trade-name and given in brackets.

of active

Infected

Water (control) Antagonist Antagonist, autoclaved Antagonist + NYD-broth Culture filtrate

berries

(%)

L47

L66

70 a 9b 62 a 21 b 58 a

70 17 68 23 79

Within each column values followed not significantly different according range test (P10.01).

a b a b a

by the same letter were to Duncan’s multiple

Isolates L47 and L66 grew in culture up to 33°C; higher temperatures were lethal. Lack of growth at temperatures over 33°C which is conTable 2 Effect of A. pullulans L47 and C. oleophila L66 on B. cinerea spore germination in the presence of low and high nutrient concentrations Medium

Water

Spore germination after 24 h 0

(control)

Wash water of: Unwounded grape Unwounded grape Unwounded grape Wounded grape luted) Wounded grape luted) + L47 Wounded grape luted) + L66 Wounded Wounded Wounded

grape grape grape

(%)

berries berries + L47 berries+L66

8a 0 c 2 b

berries

(50% di-

60 a

berries

(50% di-

33 b

berries

(50% di-

26 c

berries berries + L47 berries + L66

Wounded grape berries+ 10% grape juice Wounded grape berries+ 10% grape juice + L47 Wounded grape berries + 10% grape juice + L66

83 a 50 c 64 b 100 a 100 a 100 a

Within each group values followed by the same letter were not significantly different according to Duncan’s multiple range test (P50.01).

the percentage

ingredient

are

sidered usually suitable for the development of human mycosis (Hurley et al., 1987), can be considered as a positive characteristic. Occasionally, the yeast-like fungi have been reported as human pathogens, but intensive studies have demonstrated the independence of the saprophytic strains found on plant tissues from the strains reported to be pathogenic to man (Cooke, 1959); moreover, a feeding study with A. pullulans cells did not show pathogenicity to growing rats (Han et al., 1976). The slight phytopathogenicity of isolate L47, observed on a small number of stored strawberries harvested after the optimal maturity, seems of no practical significance, although a weak phytopathogenic activity for this micro-organism under certain conditions has been reported (Vercesi et al., 1982). Of greater importance is that isolate L47 has induced a high suppression of B. cinerea sporulation. This feature may be very useful in limiting the inoculum dispersion from infected fruits to healthy ones. The activity of isolate L47 was greater than that of vinclozolin, a very effective fungicide frequently employed for the control of Botrytis rot. Since this rot originates mainly from the latent infections of floral parts, the application of the antagonists at the flower stage seems the best strategy for an effective control of Botrytis decay as found also by Bhatt and Vaughan (1962). Fruit treatment immediately prior to harvest can be

useful for protection against Rhizopus rot, especially when the weather conditions are favourable for the development of the pathogen. Isolate L47 has also shown effectiveness against Botrytis storage rot on kiwifruit when applied alone or in combination with curing, and on table grapes when applied in the field (Lima et al., 1995). The resistance of isolates L47 and L66 to some of the fungicides commonly utilised on fruits and vegetables, may be useful in their possible use in integrated control programs. Furthermore, A. pullulms may be produced on an inexpensive growth medium (Han et al.. 1976).

Acknowledgements Thanks are expressed to Mr V. Maurantonio for his technical assistance. The paper is part of a poster presented at the XIII International Plant Protection Congress. The Hague, The Netherlands, 2-7 July 1995, Abstr. no. 1250. Research supported by a Grant of the National Research Council of Italy, Special project RAISA. Subproject 4. Paper n”. 2650.

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