Postharvest biological control of grey mould (Botrytis cinerea Pers.: Fr.) on fresh-market tomatoes with Bacillus amyloliquefaciens

PII:SO261-2194(96)00042-7

Postharvest biological control of grey mould (Botrytis cinerea Pers.: Fr.) on fresh-market tomatoes with Bacillus amyloliquefaciens M. Mari*, M. Guizzardi, Criof - Universii2

M. Brunelli

di Bologna,

and A. Folchi

K Filippo Re 8, 40126,

Bologna, Italy

Antagonistic bacteria were evaluated for their effectiveness in postharvest control of grey mould on fresh-market tomatoes. Grey mould was reduced in fresh-market tomatoes treated with antagonists and artificially inoculated with Botrytis cinerea, and stored at 20°C for at least 7 days. One strain, 5PVB (Raciltus amyfoliquefaciens) was particularly effective. This strain apparently did not produce extracellular antibiotic substances, yet was highly active against the pathogen on both mature-green and red tomatoes. Treatment with 5PVB before storage at 10°C showed only fungistatic activity against grey mould. Chilling-injured mature-green tomatoes were more susceptible to B. cinerea. On chilled fruits the bacterial antagonist completely controlled the pathogen if the treatment was applied immediately after storage at 2°C. In fermentation tests on four industrial waste materials, B. amyloliquefaciens grew well and maintained its antagonistic activity when grown in two of them. In pilot experiments with wounded fruits dipped in the bacterial cell and pathogen suspension, the development of decay was effectively inhibited. Copyright @ 1996 Elsevier Science Ltd Keywords:

postharvest

biological diseases

control;

Bacillus

Fresh-market tomatoes occupy an important position in produce markets, in Mediterranean and other countries. Harvesting of mature-green tomatoes often does not coincide with commercial requirements, nor with physiological ones (Pratella and Mari, 1990). As a consequence, it is often desirable to hold picked unripe tomatoes for a few days before marketing in cool-storage (Hobson, 1981). However, storage of mature-green tomato fruits is difficult because of their susceptibility to chilling in.jury at temperatures below lO”C, and to increased mtcrobial decay (Marangoni and Stanley, 1991). Alternaria rot and grey mould rot (Botrytis cinerea Pers.) are the most widespread microbial infections. Grey mould is the most important pre-harvest and postharvest disease on fresh-market tomatoes (Chastagner and Ogawa, 1979). Preharvest chemical control is not effective because about half the infections originate at points where the fruit is in contact with the soil (Chastagner, Ogawa, Manji and Petrie, 1977). No fungicides have yet been registered for postharvest treatment of B. cinerea in tomatoes. Research has therefore concentrated on alternative methods. Heat treatment with heated air or water has been shown to contribute to the control of B. cinerea. In fact, holding mature-green tomatoes at 38°C for 3 days completely inhibits pathogen growth (Fallik, Klein, Lomaniec, Lurie and Lalazar, 1993). Griberg,

‘To whom correspondence

should be addressed.

amyloliquefaciens;

Botrytis

cinerea;

tomato

fruits;

In California, postharvest treatments include dipping mature-green fruits in heated (3842°C) chlorinated water, followed by a fresh-water or chlorinated water spray rinse and application of a wax containing 2.5% ortho-phenylphenol (Hoy, 1984). Application of ultraviolet-C light (UV-C, 254 nm) at a range of dosages (1.3-40 KJ mm’) reduces the infections caused by B. cinerea on artificially inoculated tomatoes (Liu, Stevens, Khan, Lu, Wilson, Adeyeye, Kabwe, Pusey, Chalutz, Sultana and Droby, 1993). Chitosane (produced by deacetylation of chitin) has fungicidal activity against B. cinerea on mature-green tomatoes, and it may be used as a surface film to regulate gas exchange and moisture loss (Wilson, El Ghaouth, Chalutz, Droby, Stevens, Lu, Khan and Arul. 1994). Antagonistic micro-organisms (bacteria, yeasts, moulds) have also been the object ofconsiderable research as an alternative to synthetic fungicides effective in the control of postharvest diseases (Wilson and Wisniewski, 1994). Biological control experiments with grey mould on fresh-market tomatoes have given poor results: such experiments have mainly involved the preharvest phase. Elad, Kohl and Fokkema (1994), screened 60 isolates of saprophytic microorganisms, and found that several of the bacteria and fungi reduced conidia germination of B. cinerea and controlled grey mould disease in tomatoes. The antagonists are normally isolated by screening epiphytic microflora of phyllosphere or rhizosphere (Smilanick, 1994). Few biocontrol investigations of

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Postharvest control of Botrytis cinerea: M. Mari et al.

endophytic bacteria within tissues of healthy fruits have been reported thus far (Pratella, Mari, Guizzardi and Folchi, 1993). This has led to the suggestion that this special endophytic microbial flora, capable of living and reproducing within the fruits, constitutes a viable source of antagonist micro-organisms for inhibiting wound-infecting pathogens. The aim of this study was to investigate the possibility of postharvest biological control of B. cinerea on fresh-market tomato fruits, using these special endophytic micro-organisms.

Materials

and methods

Pathogen inoculum

A strain of B. cinerea isolated from tomato fruit was used in the experiments. The pathogen was maintained on malt extract agar (MEA). Conidial formation was induced on oat meal agar (OMA: 60 g oat meal, 10 g sodium nitrate, 30 g saccharose and 12 g agar per 1000 ml of distilled water). The culture was incubated at 25°C under UV light for 12 h daily. The conidia from the monoconidial cultures were suspended in sterile water and their concentration was adjusted by dilution to lo4 conidia ml-‘. Fruits Mature-green or red tomatoes (Lycopersicum esculentum Mill.) were used, depending on the test. The

fruits were uniform in size and colour, free from wounds and rot and stored (overnight) at 10°C until use. Before each test, the fruits were washed with a sodium hypochlorite solution (1% active chlorine), then rinsed in distilled water and left to dry at room temperature. The fruits were wounded with a sharp instrument to a depth of 3 mm (3 X 3 mm) at the equator. Antagonistic bacteria

In a previous study (Pratella et al., 1993) a total of 175 endophytic bacteria were isolated from the subepidermis of various horticultural sources (cucumber, eggplant, pepper, tomato, zucchini, apricot, peach and plum). Bacteria strains were purified and kept on nutrient yeast dextrose agar (NYDA: 8 g of Oxoid nutrient broth, 5 g of Difco yeast extract, 10 g of Oxoid dextrose and 15 g of Oxoid technical agar No. 3 per 1000 ml of distilled water). In order to evaluate antagonistic activity against B. cinerea on tomato fruits, all bacterial strains were grown for 24 h on NYDA. Two loops of about 10 ~1 each were taken from the resulting colonies and diluted in phosphate buffer (50 mM at pH 6.5). The number of colony forming units (c.f.u.) was determined by dilution platingandrangedfrom9.8X107t01.9X108 c.f.u. ml-‘. Twenty microlitres of the bacterial suspensions were introduced into the wounded fruits prepared as described. After 60 min, the same fruits were inoculated in the wound with 20 ~1 of the conidial suspension of the pathogen (lo4 conidia ml-‘). Fruits were then stored at 2O”C, and 95% r.h. Rot incidence (%) and lesion diameter (mm) were recorded after 7 days. The control comprised fruits treated only with the buffer solution and subsequently inoculated with the pathogen.

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The initial evaluation was made on only 20 fruits. Those antagonists capable of reducing rot incidence by over 90% were re-screened using 30 fruits divided into three replications of 10 fruits each. Identification of endophytic bacteria

The effective biocontrol strains were selected for identification. The isolates were subjected to first stage diagnostic tests including Gram stain, KOH, aminopeptidase, catalase, anaerobic growth and oxidase tests (Schaad, 1988). In addition the bacteria were examined for endospores according to the method of Schaeffer and Fulton (1933). One strain, SPVB, was chosen for identification using the Analytical Profile Index (API) system of the Delft University of Technology. Antibiotic activity Bacterium strains (SPVB) that exhibited maximum control in preliminary tests were employed to assess

antibiotic production using a procedure similar to that of Pusey and Wilson (1984). For this purpose, bacterial culture was transferred to NYDA and incubated for 24 h at 30°C. Two loops of about 10 ~1 each were taken from the resulting colonies and transferred to 100 ml of nutrient yeast dextrose broth (NYDB) (same ingredients as NYDA, but without agar) and shake-cultured (150 r.p.m.) for 24 h at 30°C. The bacterial cells were harvested by centrifugation (10 000 g for 20 min) and the supernatant medium was filtered through a 0.22 pmpore membrane (Millipore Corporation, Bedford, MA) to ensure sterility. Tomato fruits were then treated with a cell suspension (20 ~1 of lo* c.f.u. ml-‘) or the sterile filtrate. In addition to a buffer control, fruits were treated with fresh NYDB medium. All fruits were subsequently inoculated with the pathogen as described above, stored at 2O”C, then evaluated after 7 days. Twenty fruits per treatment, divided in four replications of five fruits each, were used. The experiment was repeated twice.

Influence of ripening and storage temperature on the effectiveness of strain 5PVB

The effectiveness of the 5PVB strain against B. cinerea was assessed on both tomatoes at low temperatures and tomatoes exposed to chilling injury. Three tests were designed to investigate this relationship: Fruit ripeness.

Tomatoes at ripening - mature-green and previously described, treated suspension (10’ c.f.u. ml-‘), with an additional 20 ~1of the conidia ml-‘). The fruits were storage at 20°C.

two different stages of red - were wounded as with 20 ~1 of antagonist and 1 h later inoculated pathogen suspension (lo4 evaluated after 7 days of

Low temperature storage. Mature-green tomatoes were wounded, inoculated and treated as described in the previous test, and divided into two groups. The fruits were stored at 20” and 10°C for 7 days. After this period, all fruits were transferred to storage at 20°C and evaluated after 7 and 13 days.

Postharvest control of Bottytis cinerea: M. Mari et al. Chilling stress. This test was designed to evaluate the effectiveness of the antagonist strain SPVB on chillingstressed tomatoes. Mature-green tomatoes were stored for various periods (0, 3, 7, 10 and 14 days) at 2°C. After the chilling, all fruits were wounded, treated and inoculated using the procedures previously described, then stored at 20°C for 7 days, after which they were examined for incidence of the disease. In all three tests, the control comprised fruits treated with 20 ul of buffer solution, then inoculated with the pathogen. Twenty fruits per treatment were used, divided in four replications of five fruits each. The experiment was repeated twice.

cassette (Pusey, Hotchkiss, Dulmage, Baumgardner, Zehr, Reilly and Wilson, 1988); the pellet obtained was diluted in distilled water. The mature-green tomatoes, selected and wounded as described earlier, were treated by dipping (60 s) in the bacterial suspension (10’ c.f.u. ml-‘) and, after 1 h, were inoculated by dipping in a conidial suspension of B. cinerea (lo4 conidia ml-‘). The control comprised fruits dipped in distilled water and then inoculated as described for the test group. Each test group comprised five replications of 20 fruits. The fruits were evaluated for disease incidence after storage at 20°C for 7 days. The experiment was done twice.

Influence of the medium on antagonist

Statistical

growth

In considering the feasibility of using strain SPVB in semi-commercial tests, in vitro growth of the strain was tested on the following media: stock medium per 1 L: 4.3 g sodium chloride, 1.5 g monosodium glutamate, 1.2 g vegetable extract, 1.2 g hydrogenated vegetable fat, 1 g yeast extract, 0.34 g mixed dehydrated vegetables, 0.2 g meat extract, 0.2 g saccharose, 0.1 g flavour agents, 0.01 g spices; soya milk; tomato broth per 1 L: 200 g tomato puree, 1 g sodium chloride; and potato broth per 1 L: 200 g potato, 20 g dextrose. These materials were preferred in place of NYDB because of their lower cost. Each medium was tested in three Erlenmeyer flasks each containing 100 ml of the medium, seeded with a bacterial suspension (lo* c.f.u. ml-‘). The control comprised three Erlenmeyer flasks containing NYDB. The flasks were shakecultured (150 r.p.m.) at 30°C. Bacterial growth was measured after 24 and 48 h on each medium (1 ml for each replications). Serial lo-fold dilutions were made in phosphate buffer, and 0.1 ml of each dilution was plated on NYDA medium in triplicate. Plates were incubated at 30°C for 24 h, and the colonies estimated by performing a vital cell count (c.f.u.). A bacterial suspension (10” c.f.u. ml-‘) from every medium, obtained by centrifugation, was tested on wounded mature-green tomatoes as previously described. The fruits were examined for incidence of the disease after storage at 2O”C, for 7 days. Twenty fruits per treatment were used, divided in four replications of five fruits each. The experiment was repeated twice. Pilot test for commercial production application of strain 5PVE3

and

Bacterial cells were obtained by growing strain SPVB in a 3-litre bench-top fermentor (Bioferm, Claind, CO, Italy). A 2-ml sample of the bacterial suspension (IO- c.f.u. ml-‘) was added to 2 L of the test medium in the 3 L fermentor. Fermentation conditions were: aeration, 0.666 volume of air per volume of medium per mitt; agitation, 300 r.p.m.; temperature 25°C. Due to the vigorous and persistent foaming associated with the culture, 3 ml of an antifoam agent (Antifoam 204, Sigma Chemical Company, St Louis, MO) was added. Two growth media were used for the production of bacterial cells (5PVB):NYDB and potato broth. At the end of the fermentation process, the cultures were concentrated with a Pellicon Cassette filtration system (Millipore) with a 100 000 molecular weight filter

analysis

Statistical analyses were done with SYSTAT 5.2 (Wilkinson, 1990). Before analysis of data the homogeneity of variances was tested using Barlett’s test (Camussi, Moller, Ottaviano and Sari Gorla, 1990). All data on percentage of infected fruits or on lesion diameter (Figure 2) were subjected to the analysis of variance (one way ANOVA). Table 2 shows the data from two-way ANOVA (storage for 7 or 13 days per treatment). Mean separations were performed using the least significant difference (LSD) test, P = 0.05. A complete randomized block design was used in each test.

Results Antagonist bacteria

effectiveness

and identification

of

Among the 175 strains tested, 13 (7.4%) were active against the pathogen and reduced the percentage of fruits infected after 7 days of storage at 2O”C, by more than 50%. The strain SPVB, isolated from the internal tissues of green pepper, was used for further trials since it totally controlled the growth of B. cinerea. Tdentification tests showed that bacteria were Gram positive and produced endospores, although some isolates were more prolific in producing endospores than others. The strains were KOH and aminopeptidase negative, and catalase and oxidase positive, they did not grow in anaerobic conditions and belonged to the genus Bacillus. The API system identified the strain SPVB as B. amyloliquefaciens. Antibiotic

activity

The highest level of grey mould control was obtained with bacterial cells, resuspended in buffer solution, which reduced disease incidence by more than 90% when compared to the control (only buffer solution) (Table I). The cell-free filtrate was completely ineffective. Influence of fruit ripeness temperature on biological

and of storage control efficacy

Fruit ripeness. The stage of fruit ripeness influenced the development of B. cinerea. After 7 days storage at 2o”C, the red-tomato control group had 95% incidence of rot, which was significantly higher (P < 0.0001) than the green-tomato control group (25%). However at the

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second inspection, after 10 days of storage, rot incidence was 100% and 75%, respectively. Strain SPVB completely inhibited the growth of B. cinerea both on mature-green tomatoes and on red tomatoes, after 7 and 10 days of storage at 20°C (Figure I). Low temperature storage. When the bacterium was introduced into the wounds before storage, the temperature of fruit storage influenced antagonist activity. At lO”C, strain 5PVB was capable of significant (P < 0.0001) fungistatic activity, inhibiting the pathogen more effectively (P = 0.0008) after 7 days (6.7% infected fruit) than after 13 days (78.3% infected fruit) (Table 2). Reduction of infection rate was nonetheless significant, with respect to the control, after both 7 and 13 days. Chilling stress. Storage of the tomatoes at a critical temperature (2°C) influenced the diameter of the lesions produced by B. cinerea (Figure 2). Tomatoes stored for 14 days at 2”C, and 5 days at 20°C had significantly (P = 0.01) larger lesions (59 mm) than those stored only at 20°C (36 mm). Chilling injury does not seem to influence the effectiveness of biological control if the antagonist is introduced at the end of storage. In fact, strain SPVB controlled B. cinerea after 14 days’ storage at 2°C and an additional 5 days’ storage at 20°C (Table 3).

b

0

on fresh-market tomato fruits with strain 5PVB of Bacillus

amyloliquefaciens TreatmentY Buffer NYDB Bacterial Cell-free

Infected

fruits 1%)

83.3 66.7 6.7 66.3

cells filtrate

a’ a b a

“Buffer solution or NYDB (nutrient-yeast-dextrose broth) were used as control. Bacterial cells were harvested by centrifugation. Cell-free filtrate was obtained by passing supernatant through a 0.22 pm-pore membrane filter. Each value is a mean of four replicates of five fruits. The experiment was repeated twice ‘Percent of fruits with decay after 7 days at 20°C. Means followed by the same letter do not differ significantly according to LSD test (P = 0.05)

loo,-

I

Figure 2. Influence of temperature on the susceptibility of mature-green tomatoes to Botrytis cinerea. Effect on size of lesions of holding at 2”C, for 0 to 14 days, before inoculation with pathogen (tomatoes held an additional 5 days at 20°C after inoculation). Each value is a mean of 20 fruits, data were pooled from two experiments. Bars with the same letter did not differ significantly at P = 0.05 by LSD test

Table 2. Effect of storage temperature on biological control of Botrytis cinerea on mature-green tomatoes after 7 and 13 days, with Bacillus amyloliquefaciens (5PVB) Infected fruits (%)Y Treatments

10°C

20°C 7 days

13 days

7 days

13 days

53.3 b’ 0.0 c

73.3 a 0.0 c

93.3 ab 6.7 c

100.0 a 78.3 b

YFruits wounded and challenged with bacterium and/or pathogen were stored at 20°C or 10°C for 7 days. Twenty fruits per treatment were used. data were pooled from two experiments. Percentage of fruits with decay was calculated after 7 and I3 additional days at 20°C ‘For each temperature, means followed by the same letter do not differ significantly according to LSD test (P = 0.05)

Table 3. Effect of chilling injury on biological control with Bacillus amyloliquefaciens (SPVB) against Botrytis cinerea on mature-green tomatoes

Treatments Control 5PVB

0 93 a Ob

Infected fruits (%)’ Days at 2°C 3 10 7 100 a Ob

100 a 7b

Influence of the medium on the growth effectiveness of strain 5PVB

”

Control

5PVB

Mature-green tomatoes

Figure 1. Effect of tomato fruit maturity on biological control of Bottytis cinerea with Bacillus amyloliquefaciens (5PVB). Mature green and red tomatoes were wounded and challenged with 5PVB and/or pathogen. After 7 days at 20°C the infected wounds (%) were recorded. Twenty fruits per treatment were used and data were pooled from two experiments. After 10 days, bars with same letter did not differ significantly at P = 0.05 by LSD test

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100 a 7b

I4 93 a Ob

‘After storage at 2°C the fruits were ino’culated with pathogen, treated with bacterium and kept at 20°C for 7 days. Twenty fruits per treatment were used and data were pooled from two experiments. Means in a column. followed by the same letter do not differ significantly at P = 0.05 by LSD test

IO days

Control SPVB Red tomatoes

I4

10

Days at 2’C

Control 5PVB Table 1. Inhibition of decay caused by Bottytis cinerea

3

8

and

After 24 h at 30°C the growth of the bacterial cells ranged from less than lo” c.f.u. ml-’ in tomato broth to 7.3 X 10’ c.f.u. ml-‘insoyamilk.Inthecontrol(NYDB) bacteria concentration was 5.7 X lo8 c.f.u. ml-‘. The tomato broth proved to be the exception, probably due to its low pH value (4.47), showing oor growth after both 24 and 48 h (2.4 X 10” c.f.u. ml- P). After 48 h, the bacterial cell concentration in the remaining media were similar, with the exception of the soya milk medium, which was significantly higher (1.3 X 10” c.f.u. ml-‘) (Table 4).

Postharvest Table 4. Growth of Bacillus amyloliquefaciens (5PVB) on nutrient viva antagonistic activity against Bofrytis cinerea oil tomato fruits

yeast dextrose

control

of Bottytis

broth (NYDB) or industrial

Cell counts (c.f.u. ml~~‘)Y Growth

medium

after 24 h

‘Bactenum NYDA ‘/,I

WV

ceil concentration

was calculated

(nutrient-ye:rst-dextrose bioassay

treatment significantly

agar)

of antagonistic

wcr~’ used.

data

at I’ = Il.05

activity

were

by I.SD

in samples

+ standard

pooled

two

medium

after

f f f f ir

3.8 2.8 1.4 1.2 2.9

materials

lox 10’ 10’ IO’ 10’

24 and 48 h of fermentation.

and its in

15 b oc 100 a 100 a IO bc each value

is a mean of three

separate

replicates

on

error

of bacterium from

of the growth

2.2 2.4 1.3 2.4 2.2

waste

M. Mari et a/.

Infected fruits (%)’ after 8 days at 20°C

after 48 h

s.7 t 2.6 IOX 4.5 f 2.3 IOX 7.3 * 6.2 IO”
NYDB Potato dextrose broth Soya milk Tomato juice Stock

cinerea:

preparation

experiments.

against Mean

6.

cinerecr was performed

of infected

fruits

as described

of the control

was 100%.

under

Materials

Means

followed

and methods.

Twenty

by the same letter

fruits

per

do not differ

test

Discussion

NYDB

PDB

Figure 3. Effect of dip application of Bacillus amyloliquefaciens (5PVB) grown on different media (NYDB and PDB) on the incidence of postharvest decay (grey mould) in tomato fruits. Tomato fruits were wounded, treated by dipping in the bacterial suspension (10’ cell ml-‘) and inoculated, again by dipping, with Bofryfis cinerea (lo4 conidia ml-‘). After 7 days at 20°C the infected wounds (%) were recorded. One hundred fruits per treatment were used and the experiment was repeated twice. Bars within a medium with same letter did not differ significantly at P = 0.05 by LSD test

The medium influenced the effectiveness of the 5PVB strain against B. cinerea. The best activity was had with the bacterium grown on PDB (0% infected fruits); results were also good with stock medium (10% infected fruits) and NYDB (15% infected fruits) (Table 4). The degree of control obtained with soya milk and tomato broth was null. In the former case, the procedures necessary for harvesting the bacterial cells (centrifugation or filtration) probably caused an accumulation, in the pellets, of the lipid compounds present in suspension in the soya milk, interfering with a correct bacterial cell count. In the latter case, over 48 h, a cell concentration sufficient for B. cinerea control was not reached. Pilot test for commercial application of SPVB

production

and

The fermentation process, carried out with a bench-top fermentor on different substrates (PDB, NYDB), produced a considerable quantity of bacterial cells, suitable for immersion treatment with a high number of fruits. The treatments carried out with 5PVB bacterial cells from the PDB medium significantly (P < 0.0001) with respect to the waterreduced rotting (36.2%) treated control (94.2%). A similar pattern was observed with NYDB medium (Figure 3).

Control of grey mould using epiphytic antagonists has been successful for pre- and postharvest treatments of various crops: strawberries (Bhatt and Vaughan, 1962), grapes (McLaughlin, Wilson, Droby, Ben-Arie and Chalutz, 1992) apples (Janisiewicz and Roitman, 1988; Roberts, 1990; Tronsmo, 1991), and beans and tomatoes (Elad et af., 1994). A bacterial population is normally present inside the healthy tissues of fruits and vegetables (Samish, Etinger-Tulczynska and Bick, 1963). The isolated endophyte population was tested on freshmarket tomatoes for its effectiveness against B. cinerea, and about 7% of the strains selected satisfactorily controlled the pathogen. Recently, endophytic bacteria have also been used for controlling Monilinia luxa (Aderh. & Ruhl. Honey) and Rhizopus stolonifer (Ehrenb.: Fr.) Vuill. (Pratella et al., 1993). To this evidence, we have added a strain, SPVB, identified as B. amyloliquefaciens, which controls B. cinerea on fresh-market tomato fruits. It has long been recognized that susceptibility of tomatoes to decay is increased if they are held for a week or more at low temperatures (McColloch and Worthington, 1952). Grey mould is one of the most prevalent sources of decay under such conditions. In fact, the direct relationship between increased susceptibility to rot and fruit storage at low temperature (McColloch, 1951) has long been recognized, especially as related to the degree of ripening (Dennis, 1983). The ripeness of the fruit influences the development of B. cinerea, which is slower in mature-green tomatoes than in red tomatoes. Glyco-alkaloid tomatine could be responsible for this. The tomatine content is high in green fruits and is absent in red, ripe fruits (Verhoeff and Liem, 1975). Nevertheless, at either stage of maturity, the strain 5PVB provided satisfactory control of the pathogen. B. amylofiquefuciens (SPVB) had a fungistatic effect on B. cinerea in mature-green tomatoes stored at low temperature (IO’C) and significantly reduced pathogen growth during the first 7 days of storage. However, storage of tomatoes at temperatures below 10°C for more than 24 h must be avoided because various symptoms of chilling injury may seriously impair the market quality of the fruit (Ryall and Lipton, 1972). Optimum temperatures for short-term storage and transport are, for mature-green tomatoes, 12.8 to 15,6”C(Kasmire, 1985). Although tomatoeswithchilling

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Postharvest control of Sor@s cinerea: M. Mari et al. injury are more susceptible to attack by B. cinerea, the antagonist B. amyloliquefaciens is capable of stopping development of the pathogen, if the fruits are treated immediately after storage at 2°C. The results of the in vitro test show that the strain SPVB produced a considerable quantity of bacterial cells in three waste materials (PDB, soya milk and stock) as compared with fermentation in NYDB. The in viva activity of antagonistic bacterium proved to be too low for soya milk and tomato broth. In both cases, sufficient concentrations for biological control (10’ c.f.u. ml-‘) were not attained. The use of soya milk defatted medium (Dulmage, 1971) may improve the quantity of bacterial cells harvested. In semicommercial tests, it is important that the antagonist grow in industrial waste materials without altering its antagonistic effectiveness. The positive results of large-scale experiments and pilot tests using B. subtilis (B-3) against peach brown rot (Pusey et al., 1988) and Pi&a guillermondii (US-7) versus P. digitatum of citrus fruit (Droby, Hofstein, Wilson, Wisniewski, Fridlender, Cohen, Weiss, Daus, Timar and Chalutz, 1993) confirm the feasibility of large-scale production and application of biocontrol agents. Our results show that the commercial production and application of the SPVB strain of B. amyloliquefaciens, merits special attention and lead us to believe that the practical use of this bioproduct for postharvest control of grey mould rot on tomatoes is realistic, although it could be further improved through efforts to develop the fermentation and formulation technology.

Elad, Y., Kohl, J. and Fokkema, N. J. (1994) Control of infection and sporulation of Botrytis cinereu on bean and tomato by saprophytic bacteria and fungi. Europ. J. Plant Puthol. 100, 315-336 Fallik, E., Klein, J., Griberg, S., Lomaniec, E., Lurie, S. and Lalazar, A. (1993) Effect of postharvest heat treatment of tomatoes on fruit ripening and decay caused by Botrytis cinerea. Plant Dis. 77, 985-988 Hobson, G. E. (1981) The short-term Sci. 56, 363-368

storage of tomato fruit. J. Hort.

Hoy, M. W. (1984) Toxicity of the surfactant Nacconol to four decaycausing fungi of fresh-market tomatoes. Plant Dis. 68, 699-703 W. J. and Roitman, J. (1988) Biological control of blue mould and grey mould on apple and pear with Pseudomonas cepaciu. Plant Dis. 78, 1697-1700

Janisiewicz,

Kasmire, R. F. (1985) Postharvest handling systems: Fruit vegetables. In Postharvest Technology of Horticultural Crops (Ed. by A. A.

Kader, R.F. Kasmire, E. Gordon Mitchell, u. S. Reid, N. E. Sommer and J.F. Thomosonl Universitv of California. Div. Agriculture and Natural Re’sourkes, OakIan& pp. 139-142 Liu, J., Stevens, C., Khan, V. A., Lu, J. Y., Wilson, C. L., Adeyeye, O., Kabwe, M. K., Pusey, P. L., Chalutz, E., Sultana, T. and Droby, S. (1993) Application of ultraviolet-C light on storage rots and ripening of tomatoes. J. Food Prot. 56, 868-873 A. G. and Stanley, D. W. (1991) Studies on the longterm storage of mature, green tomato fruit. J. Hort. Sci. 66, 81-84

Marangoni,

McColloch, L. P. (1951) Alternaria tenuis the cause of tomato fruit rot. US Dept. Agr., Plant Dis. Reptr. 35, 234-236 McCollocb,

L. P. and Worthington,

J. T. (1952) Low temperature as tomatoes to Alternariu

a factor in the susceptibility of mature-green rot. Phytopathology 42, 425-427 McLaughlin, R. J., Chalutz, E. (1992)

Wilson,

C. L.,

Droby,

S.,

Ben-Arie,

R. and

Biological control of postharvest. diseases of grape, peach and apple with the yeast Kloeckera apiculata and Candida guilliermondii.

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G. C. and Mari, M. (1990) Biologia e tecnica post-raccolta della melanzana, peperone e pomodoro. In: Prodotti Ortivi al10 Stat0 Fresco: Biologia e Tecniche Post-Raccolta. Cesena, Italy, 4-23

Pratella,

Acknowledgements

Pratella,

Research supported by National Research Council of Italy, Special project RAISA, Sub-project 4, Paper No. 2627.

G. C.,

Mari,

M.,

Guizzardi,

M. and Folchi,

A. (1993)

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