Directory UMM :Data Elmu:jurnal:P:Postharvest Biology and Technology:Vol20.Issue1.Aug2000:

Postharvest Biology and Technology 20 (2000) 99 – 106

Evaluation of the fungicidal properties of plant extracts to

reduce

Rhizopus stolonifer

of ‘ciruela’ fruit (Spondias

purpurea

L.) during storage

S. Bautista-Ban˜os

a,

_{*, M. Herna´ndez-Lo´pez}

b

_{, J.C. Dı´az-Pe´rez}

c

_,

C.F. Cano-Ochoa

b

a_{Centro de Desarrollo de Productos Bio´ticos,Carr. Yautepec-Jojutla km,8.5,San Isidro Yautepec Mor, Mexico 62731}

b_{Instituto Tecnolo´gico de Zacatepec,Apdo. Postal 45,Mor, Mexico}

c_{Coastal Plain Experiment Station,Department of Horticulture,Uni6ersity of Georgia,Tifton,GA 31793-0748, USA}

Received 24 April 1999; accepted 23 April 2000

Abstract

Rhizopus stoloniferis one of the main postharvest pathogens of ‘ciruela’ fruit (red mombin) (Spondias purpureaL.) during handling and storage. To evaluate the fungicidal potential of plants indigenous to the state of Morelos, Mexico, aqueous extracts of leaves or stems of 19 different plant species were tested againstRhizopus development in vitro and in vivo. Extracts were applied to fruit of three botanical varieties of ciruela: fruit skin turning green to red, green to yellow or green to orange, grown throughout the year over dry and wet seasons. In vitro evaluations were carried out to observe mycelial growth, sporulation and conidial germination. Evaluations on fruit were percentage and disease severity, soluble solids content (SSC) and weight loss after 4 days storage at ambient temperature. In general, leaf extracts had better fungicidal effects than stem extracts. For in vitro studies, leaf extracts inhibited sporulation and spore germination more than mycelial development. Leaf extracts ofAnnona cherimola,

Bromelia hemisphaericaandCarica papayainhibitedRhizopussporulation and rot development on the yellow variety whereas extracts ofCasimiroa edulisreducedR.stoloniferrot on red ciruela. Infection spread from 25 to 100% of the fruit surface depending on extract or ciruela variety. After storage, SSC values were not significantly different. Less weight loss was observed for the orange variety than the other varieties. Further studies need to be undertaken to isolate the active compounds from those extracts with fungicidal potential. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Red mombin; Yellow mombin; Biofungicides; Postharvest rots; Aqueous extracts

www.elsevier.com/locate/postharvbio

1. Introduction

The ‘ciruela’ (jocote or red mombin) is a widely distributed tropical tree in Mexico. A wide variety and forms of seedling races can be found in this

* Corresponding author.

S.Bautista-Ban˜os et al./Posthar6est Biology and Technology20 (2000) 99 – 106

100

country (Popenoe, 1974). The state of Morelos, Mexico is important for production of this fruit. Ciruelas are consumed mainly as fresh produce (Niembro, 1986). Fruit size, shape and colour can be different according to the botanical variety and ripening stage (Popenoe, 1979; Kersul et al., 1998). Production of these fruit takes place throughout the year. The main botanical variety of ciruela (red mombin) is produced from Septem-ber to OctoSeptem-ber. However other botanical varieties

ofSpondias purpureaare harvested during the dry

season (February – May) and the beginning of the wet season (June – July) (Leon and Shaw, 1990).

Ciruelas are rarely sent to distant markets be-cause of their high perishability (Dı´az-Pe´rez et al., 1998) and incidence of postharvest pathogens (Bautista et al., 1997). Rots caused by Rhizopus

stolonifer can account for more than 50% of

postharvest losses during fruit handling and stor-age. Fruit growers from this region cannot afford the high cost of fungicides, yet, traditionally they have used botanical species as a source of medicines, biocides and other materials whose fungicidal properties are still unknown (Sarukha´n, 1995).

The aim of our study was to investigate the fungicidal potential of various plant species to reduceR.stoloniferof three botanical varieties of ciruela fruit and to evaluate their effect on fruit quality after ambient storage.

2. Materials and methods

2.1. Plant material and fungal cultures

Plant material was collected within the state of

Morelos. Except for Bromelia hemisphaerica L.,

the species collected consisted of leaves and stems

of Achras sapota L., Annona cherimola M., An

-nona reticulata L., Annona muricata L., Arc

-tostaphylos polifolia, H.B.K., Carica papaya L.,

Crataegus mexicana Moc et Sess., Casimiroa

edulisLlav. et Lex.,Citrus limonL.,Crysophyllum

cainito L., Dyospiros ebenaster Retz., Inga spuria

Humb. et Bompl.,Persea americanaMiller,Pithe

-cellobium dulce Benth., Pouteria sapota Jacq.,

Prunus capuliCav.,Psidium guaja6aL. andSpon

-dias purpurea L. Once in the laboratory, leaves or

stems were dipped in 1% sodium hypochlorite, rinsed with distilled water, air-dried, macerated with the aid of a grinder and a blender and stored in black polyethylene bags at 20°C until further use.

R. stolonifer was isolated from rotting ciruela

and the isolated strain was maintained in PDA. To maintain pathogenicity of the fungus, periodic inoculations and reisolations from infected ciruela were carried out.

2.2. Preparation of extracts

Leaf and stem extracts were prepared following the method of Ahmad and Prasad (1995). Dry powders of leaves or stems were mixed with dis-tilled water (1:5 w/v) and left overnight. The extract was then filtered and autoclaved (103.3 kPa for 15 min). For in vitro studies, before

sterilization, extracts were incorporated into

Potato Dextrose Agar (PDA) medium (1:4 v/v)

and poured into Petri plates. One 5-mm agar disc containing R. stolonifer was placed at the centre of each plate and incubated for 4 days at 25°C.

2.3. Acti6ity of plant extracts in6itro

After 4 days incubation at 25°C, colony diame-ter, sporulation and percentage germination were evaluated. To collect conidia for sporulation and germination tests, Petri dishes from each treat-ment were rinsed with 10 ml sterile distilled water, the surface scrapped with a glass rod and the filtrate passed through cotton wool. To test spore viability, a 0.5-ml aliquot of spore suspension was placed on a 20-mm diameter agar disk and after 5

h at room temperature (2094°C) stained with

lactophenol acid fuchsin. Both, sporulation and

germination were determined at 40×

magnifica-tion. Each treatment was repeated three times.

2.4. Fruit preparation

S.Bautista-Ban˜os et al./Posthar6est Biology and Technology20 (2000) 99 – 106 101

harvested in June was turning green to yellow (‘yellow’ variety) and fruit harvested in October was turning green to orange (‘orange’ variety). Fruit were transported to the laboratory, washed in chlorinated water (200 ml l−1_{) for 5 min, rinsed}

with distilled water and air-dried. Fruit were dipped in either leaf or stem extracts for 15 min and dried. After treatment, ciruela were evenly sprayed with a spore suspension 1×108_l−1_ofR.

stolonifer. Fruit were kept in humidified chambers

for 4 days at ambient temperature. For red or yellow ciruela storage, ambient temperature was within a range of 2893°C. For orange ciruelas,

the temperature was 2092°C.

2.5. Acti6ity of plant extracts on fruits

After the ripening period, percentage infection, disease severity, SSC (hand refractometer Atago 0 – 32°) and weight loss were evaluated for 10 ciruelas per treatment.

2.6. Statistical analysis

Means separation by Tukey’s multiple range test (PB0.05) was carried out separately for leaf or stem extracts in those parameters regarding in vitro studies.

Weight loss and SSC were analysed through ANOVA. Treatments were arranged in a com-pletely randomized design, while percentage dis-ease was analysed using thex2procedure. Disease

severity was ranked 1 to 5 where 1=0% of

sur-face fruit rotten, 2=25%, 3=50%, 4=75% and

5=100%.

3. Results and discussion

In general, better fungicidal effects were ob-served with leaf extracts than with stems extracts for both in vitro and for the whole ciruela fruit studies. Compared to the other treatments stem extracts ofA.reticulatasignificantly reducedRhi

-zopus mycelial growth (PB0.05) (Table 1).

Sporulation was completely inhibited with leaf extracts of A. cherimola, A. reticulata, B. hemis

-phaerica, C. papaya, C. limon, P. dulce and P.

sapota. Conidial germination after a 5-h

incuba-tion varied according to each extract. The highest germination was observed in control medium but

Rhizopus germination did not occur in medium

containing leaf extracts of A. reticulata. Less

ger-mination was observed with leaf extracts of A.

muricata,B.hemisphaerica,C.mexicana,P.amer

-icana, P. guaja6a and S. purpurea and stem

ex-tracts of A.reticulata.

Although extracts had similar effects on the three ciruela varieties, the fungicidal effect was greater on the red or yellow varieties than on the orange ciruelas (Tables 2 – 4). For example, R.

stolonifer development was completely inhibited

in red ciruelas when dipped in leaf extracts of C.

edulis (Table 2) and in yellow ciruelas when

treated with leaf extracts of A. cherimola, B.

hemisphaerica and C. papaya (Table 3), while in

orange ciruela the lowest percentage infection was 60% with stem extracts of A. reticulata and P.

dulce (Table 4). Extracts from various plant

or-gans of C. papayahave been shown to have high

fungistatic or antiparasitic activity over human diseases andC.limonhas useful insecticidal prop-erties (Mwaiko, 1992; Osato et al., 1993; Giordani et al., 1997; Mistra et al., 1988). Extracts of C.

edulis also have a broad range of medical

activi-ties (Vidrio and Magos, 1991). Previous studies have demonstrated the antifungal activity of aqueous extracts of leaves of P. dulce to reduce

infection by Uromyces appendiculatus on bean

crops (Montes et al., 1990).

In this study, disease severity varied depending on the plant extracts used and the variety of ciruela. Infection spread up to 100% of the surface of the red or yellow ciruelas, compared with only 75% of the orange ones (Tables 2 – 4).

SSC of the treated fruit were not significantly different at each ciruela variety (data not shown). After the storage period SSC (%) ranges for red ciruela were 15.6 – 19.2, for yellow ciruela 16.2 – 19.0 and for the orange ciruela 16.6 – 19.1.

S

Effect of foliar and stem extracts on in vitro development ofR.stoloniferafter 4 days incubation at 25°C

Stemsa Leavesa

Plant species

Sporulation Conidial germination (5 h) Conidial germination

3.9ab 41bcd 24bcd 97a 96a

1.Achras sapota 4.0a

4.0a 25fg 67abc

2.Annona cherimola 3.8ab 0d –

28c

4.0a 3.6b 10g

3.Annona reticulata 3d 0d

95ab

4.0a 36bcd 14cd 4.0b 97a

4.Annona muricata

3.9a

4.0a 29bcd 27bcd 80abcd 89ab

5.Arctostaphylos

7.Carica papaya 4.0a 0d

4.0a 68abc 27bcd 83abc 77abc

8.Casimiroa edulis 4.0a

56abc

0d – 4.0a 38cdefg

3.9ab

9.Citrus limon

4.0a

3.8ab 17cd 15cd 76abcd 58abc

10.Crataegus

mexicana

20bcd 4.0a 57abcdefg 83abc

11.Chrysophylum 4.0a 40bcd

cainito

4.0a 43bcd 4.0a 62abcdef 89ab

12.Dyospiros 70ab

ebenaster

36bc

4.0a 26efg

52abc

13.Inga spuria 4.0a 77ab

4.0a 24bcd 15cd 82abc 82abc

14.Persea americana 4.0a

– 4.0a 49bcdefg 39bc

4.0a

16.Pouteria sapota 4.0a

4.0a 52abcd 47bcd 27efg 87ab

17.Prunus capuli 4.0a

18.Psidium guaja6a 4.0a 40bcd 14cd 3.9a 33defg 75abc

84abc 4.0a

9cd 74abcde

3.9ab 30bcd

19.Spondias purpurea

S

Effect of foliar and stem extracts on percentage infection, disease severity and percentage weight loss of red ciruela inoculated byR.stoloniferand kept at ambient temperature for 4 days

Plant species Leaves Stems

Percentage infection

Disease severitya _{Percentage weight loss Disease severity}a _{Percentage weight loss} Percentage infection

(PB0.001)

(PB0.001) (PB0.001) (PB0.001)

Control 80 2 14.297.3

80 2 10.292.4

1.Achras sapota 60 3 12.191.2

40 3 60 2 10.292.7

4. Annona 11.790.7

muricata

2 10.593.1

5.Arctostaphylos 50 3 14.193.3 50

polifolia

c c

c 12.491.0

6.Bromelia 80 4

hemisphaerica

100 5 b 3 14.193.0

7.Carica papaya 60

8. Casimiroa 0 1 8.894.1 70 3 9.592.4

13.792.2 60 2 13.796.4

11.Chrysophyllum 40 2

cainito

13.Inga spuria 50 3 7.493.8

70

80 3 12.291.9 4 10.893.3

14.Persea

americana

12.691.6 20 2 14.592.6

15.Phytecellobium 50 2

dulce

40 2 6.293.2 4 10.892.1

17.Prunus capuli 70

2

18.Psidium 60 3 11.490.9 80 7.794.3

guaja6a b_{Fruit of these treatments completely rotten.}

S

Effect of foliar and stem extracts on percentage infection, disease severity and percentage weight loss of yellow ciruela inoculated by R.stoloniferand kept at ambient temperature for 4 days

Leaves Stems

Plant species

Disease severitya _{Percentage weight loss} Percentage weight loss

Disease severitya _{Percentage infection} Percentage infection

(PB0.001) (PB0.01) (PB0.001) (PB0.001)

9.091.2

Control 30 2

10 2 10.194.5 3 8.795.2

1.Achras sapota 50

0 ₁ 100 5 b

5.Arctostaphylos 20 2 7.692.8 70

polifolia

7.Carica papaya 100

8.Casimiroa 10 _{2 15.19 5.7 100} 5 b

20 2 10.192.5 2 12.993.9

10.Crataegus

mexicana

10.49 2.1 30 2 10.894.6

11.Chrysophyllum 20 2

cainito

13.Inga spuria 40 3 12.696.7

80

20 2 12.598.2 4 12.794.1

14.Persea

americana

11.093.7 100 5 b

15.Phytecellobium 30 2

dulce

17.Prunus capuli 100

3

18.Psidium 10 2 10.994.4 60 4.393.9

guaja6a

5 b

9.091.8 2

19.Spondias 20 100

purpurea

a_{Disease severity: 1=0%, 2=25%, 3=50%, 4=75 and 5=100% of surface of fruit rotten.} b_{Fruit of these treatments completely rotten.}

S

Effect of foliar and stem extracts on percentage infection, disease severity and percentage weight loss of orange ciruela inoculated byR.stoloniferand kept at ambient temperature for 4 days

Plant species Leaves Stem

Percentage infection

Disease severitya _{Percentage weight loss Disease severity}a _{Percentage weight loss} Percentage infection

(PB0.001)

(PB0.001) (PB0.001) (PB0.001)

Control 90 3 2.590.9

90 3 1.891.2

1.Achras sapota 70 2 0.690.3

100 4 80 3 1.090.8

5. Arctostaphylos 90 3 1.791.3 100

polifolia

b b

b 2.091.1

6.Bromelia 100 3

hemisphaerica

100 4 1.490.7 3 0.790.3

7.Carica papaya 100

100

100 3 2.791.9 3 0.990.8

10.Crataegus

mexicana

3.492.7 100 4 2.491.0

11.Chrysphyllum 100 3

cainito

13.Inga spuria 90 4 2.592.3

80

80 3 2.792.0 2 2.191.5

14.Persea

americana

2.191.2 60 3 0.690.4

90

17.Prunus capuli 100

3

18.Psidium 100 4 2.491.5 90 2.091.6

S.Bautista-Ban˜os et al./Posthar6est Biology and Technology20 (2000) 99 – 106

106

similar total weight loss values when this variety was stored at 10°C for 7 days, with differences in daily weight loss according to harvesting location and fruit ripening stage. In this study, harvesting location and temperature were not controlled fac-tors. Probably those values of weight loss for each ciruela variety are more related to changes in the ambient temperature and the variety per se than with the effect of each extract over the fruit.

Results of this investigation demonstrated the fungicidal potential of a range of plant species. Future work will include the isolation of the active compounds of those extracts with high fungicidal properties.

Acknowledgements

This research was sponsored by Consejo Na-cional de Ciencia y Tecnologı´a (Project: 26414-B)

and Instituto Polite´cnico Nacional (Project:

COFFA, CGP1-978039).

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