ScientiaHorticulturae, 40 (1989) 105-112 105 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Postharves t Bio logica l Control of Penicil l ium Rots of Citrus w i th Antagonis t i c Yeasts and Bacter ia

CHARLES L. WILSON ~ and EDO CHALUTZ 2

USDA, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV 25430 (U.S.A.) -'Institute for Technology and Storage of Agricultural Products, ARO, The Volcani Center, Bet Dagan, 50250 (Israel)

(Accepted for publication 13 December 1988 )

ABSTRACT

Wilson, C.L. and Chalutz, E., 1989. Postharvest biological control of Penicillium rots of citrus with antagonistic yeasts and bacteria. Scientia Hortic., 40: 105-112.

Two yeasts, Debaryomyces hansenii (Zopf) Van Rij and Aureobasidium puUulans (De Bary) Arnaud, and two bacteria, Pseudomonas cepacia (Van Hall) Bergy et al. and P. syringae Burk- holder, were the most effective antagonists from over 100 isolates tested against PeniciUium dig- itatum Sacc. and Penicillium italicum Wehmer rots on citrus fruit. Overall, P. cepacia provided the best protection against both of these rots. P. cepacia produced antibiotic zones against the two PeniciUia rot organisms in culture, whereas P. syringae, D. hansenii and A. pullulans did not. All four antagonists show promise as biocontrol agents.

Keywords: Aureobasidium puUulans; biological control; citrus; Debaryomyces hansenii; Penicil- lium digitatum; Penicillium italicum; Pseudomonas cepacia; Pseudomonas syringae.

Abbreviations: ATCC = American Type Culture Collection; NYDA = nutrient yeast dextrose agar; NYDB = nutrient yeast dextrose broth; PDA = potato dextrose agar.

INTRODUCTION

PeniciUium digitatum Sacc. (green mold) and P. italicum Wehmer (blue mold) account for most decay losses of citrus worldwide (Bancroft et al., 1984). Isolates resistant to fungicides used to control these fungi also have been de- tected in the major citrus-producing areas of the world (Bancroft et al., 1984). These findings, and public pressure to reduce the use of pesticides, emphasize the need to find alternative means of controlling postharvest rot diseases of citrus.

Biological control of postharvest diseases of' fruit has recently met with re- markable success with peaches (Wilson and Pusey, 1985; Pusey eta!., 1986)

0304-4238/89/$03.50 © 1989 Elsevier Science Publishers B.V.

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and apples (Janisiewicz, 1987; Janisiewicz and Roitman, 1988). A number of antagonists for the biological control of Penicillium digitatum on citrus were evaluated by De Matos (1983), who was able to reduce mold incidence from 35 to 8% when a species of Trichoderma was inoculated with the pathogen into lemon peel. Following the earlier work of Gutter and Littauer (1953), Singh and Deverall (1984) demonstrated biocontrol with bacterial antagonists to the citrus pathogens Alternaria citri Pierce, Geotrichum candidum Link. ex Pers., and P. digitatum. Dipping wounded citrus fruit in suspensions of bacterial cells, particularly a strain of Bacillus subtilis (Ehrenber) Cohn, delayed decay by the 3 rot pathogens.

The present investigation was undertaken to find new biological-control an- tagonists of the citrus rot pathogens P. digitatum and P. italicum. We were particularly interested in finding antagonists that did not produce antibiotics as part of their mode of action. Such antagonists should gain better public acceptance since no exotic antibiotics would be introduced into the food chain if they were applied.

MATERIALS AND METHODS

Potential antagonists were isolated from the surfaces of lemons (Citrus li- mon (L.) Burro. f. ) harvested in Florida from unsprayed groves near Orlando, shipped to the USDA ARS Appalachian Fruit Research Station, Kearneys- ville, WV, and received within a 2-day period. Isolates were obtained from lemon fruit by placing individual fruit in a 600-ml beaker containing 200 ml of sterile water. The beakers containing fruit were placed on a rotary shaker at 100 rpm for 10 min. One tenth ml of the wash water was then spread on a Nutrient Yeast Dextrose Agar (NYDA) plate and allowed to incubate for 24 h before colonies were selected. The same fruit received three separate washings and the same procedures were followed. Selected colonies were then streaked three times across NYDA plates, in order to obtain colonies likely arising from a single cell. If all colonies on the plate at the final streaking looked uniform, they were assumed tobe pure; if not, they were streaked an additional three times and re-examined. All cultures were stored on silica gel in a freezer until they were tested on the fruit.

One hundred and four isolates were obtained from lemon surfaces by these procedures. A number appeared to be the same organism, but each was tested on lemon and grapefruit, Citrus paradisi (Macf.) Merrill, against Penicillium digitatum and Penicillium italicum.

Cultures of individual isolates to be tested were grown on NYDA medium for at least 3 days. A loop of the culture was then transferred to flasks contain- ing 50 ml of NYDB and incubated at room temperature on a shaker set at 175 rpm for 24 h. One ml of each seed flask was then transferred into another flask containing 50 ml of NYDB. These flasks were incubated, as before, for 48 h

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before testing them on the fruit. Cell counts, determined by dilution plating, ranged from 107 to 109 colony-forming units ml- 1. Grapefruit and lemons which had been washed in a 2% sodium hypochlorite solution and dried, were wounded 6 times with a dissecting needle. The wound size was 4-mm wide by 5-mm deep. The wounds were filled with broth containing the test organism using a small paint brush. After the wound site had dried, each wound was challenged with 20 ,ul of either P. digitatum or P. italicum at a concentration of 104 spores ml- l. After the fruit were incubated for 4 days at room temperature in covered plastic containers, to the bottom of which sterile water was added to maintain humid- ity, the number of infected wounds was counted. Wound sites were observed for up to 14 days. A minimum of 12 wounds (2 per fruit) for grapefruit and lemon was used to evaluate each isolate.

To measure further the effects of the best antagonists, antagonist/pathogen combinations were tested on individual grapefruit. The fruit was supported on Styrofoam fruit trays (Soft-Pak tray, Amoco Foam Products, Yakima, WA) in plastic containers with lids (Le Beau Products, Baraboo, WI). One wound per fruit was treated with antagonist/pathogen combinations as previously de- scribed. After incubation periods from 3 to 15 days at room temperature, the percentages of non-infected fruit were recorded. Each experiment involved 5 fruit with 2 wounds per isolate. Experiments were repeated 4 times with similar results and the data were pooled.

Preparations for viewing the four most effective antagonists with a scanning electron microscope were made by growing the isolates on NYDA over night at 30°C. Agar samples, 3 mm in diameter, with the organism were removed with a cork borer and fixed in 5% glutaraldehyde in 0.025 M phosphate buffer for 2 h. This material was then dehydrated through an ethanol series, critical point dried, sputter coated, and examined with a Cambridge Stereoscan 120 at 10K.

RESULTS

After the initial screening of 122 potential antagonists (Table 1), 4 were chosen for further testing on the basis of their antagonism against both P. digitatum and P. italicum. These 4 isolates were the ones that inhibited infec- tion either of P. digitatum or P. italicum by 50% or more 11 days after inocu- lation (=wounds infected by 50% or less). Two of the isolates were bacteria, Pseudomonas cepacia (Van Hall) Bergy et al. and Pseudomonas syringae Burk- holder, and two were yeasts, Debaryomyces hansenii (Zopf) Van Rij and Au- reobasidiumpullulans (De Bary) Arnaud. Only one of the isolates (P. cepacia) produced antibiotic zones of inhibition against P. digitatum and P. italicum on NYDA and potato dextrose agar (PDA) media. The P. cepacia isolate (Fig. lc) was isolated from soil and identified by Dr. Harvey Spurr, ARS, Oxford,

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TABLE 1

Percent wound infection of isolates 11 days after inoculation into lemon or grapefruit wounds

Isolate Identification No. 1

Percent of wounds infected

P. digitatum P. itaUcum

28 Pseudomonas cepacia (Spurr 41) 21 20 87 Debaryomyces hansenii (lemon) 46 48 40 Aureobasidiumpullulans (lemon) 42 50 64 Pseudomonas syringae (lemon) 45 64 45 Enterobacter cloacae 57 91 33 Pseudomonas maltophilia 80 45 32 Pseudomonas fluorescens 73 58 57 Klebsiella pneumoniae 70 66 19 Bacillus subtilis 83 91 79 Serratia marcesens 66 75 34 Bacillus polymyxa 100 34 16 Streptornyces noursei 100 66 15 Bacillus subtilis spp. globigii 100 58 7 Erwinia herbicola 100 75 8 Bacillus brevis 100 75

11 Pseudomonas aureofaciens 100 83 30 BaciUius cereus spp. mycoides 100 83 42 Agrobacterium tumefaciens 100 100 10 Bacillus licheniformis 100 100 9 Bacillus polymyxa 100 100

21 Bacillus thuringiensis 100 100 101 unidentified isolates above 50 above 50 Untreated control 100 100

tIsolates 8, 9, 10, 11, 15 were obtained from the USDA, ARS, Peoria, IL; Isolates 28, 30, 32, 33, 34 were obtained from Harvey Spurr, USDA, ARS, Oxford NC; and Isolate 57 was obtained from Dr. Sara Wright, USDA, ARS, Beckley, WV. A minimum of 12 wounds (2 per fruit) was used to evaluate each isolate. After inoculation, fruit were kept at room temperature for 11 days and evaluated.

NC. T h e o the r 3 isolates were i so la ted f rom l emon f ru i t wash ings a n d ident i - fied by p e r s o n n e l of the A m e r i c a n T y p e Cul tu re Collect ion, Rockvi l le , MD.

Morphological ly , D. hansenii grew unicel lular ly on solid y e a s t - m a l t agar (Fig. l a ) . In l iquid cu l ture a f t e r 1 day, smal l globose cells were obse rved m a i n l y in cha ins or c lusters , m a n y wi th one bud. Colonies were c r e a m whi te and sl ight ly raised, sh iny and round wi th s m o o t h edges. N o p s e u d o h y p h a e were observed. No ascospores were p roduced a f t e r I week on c o r n - m e a l agar, V-8 juice agar, y e a s t - m a l t agar or on ace ta te . C a rbon ass imi la t ion , n i t rogen ass imi la t ion , fer- m e n t a t i o n and v i t a m i n r e q u i r e m e n t s were typ ica l of th i s species (Rij, 1984) ( da ta no t p r e s e n t e d ) .

Aureobas id ium puUulans cells were large a n d ovoid and f o r m e d 1-4 buds

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Fig. 1. Scanning electron micrographs of the four most effective antagonists. (a) Debaryomyces hansenii; (b)AureobasidiumpuUulans; (c)Pseudomonas cepacia; (d)Pseudomonas syringae. Microns indicated above the bars.

after 2 days in liquid yeast-malt broth (Fig. lb). Growth on solid agar was filamentous with blastospores. Colonies were cream colored, turning pink to orange after 1 week. The colonies were also shiny, slightly raised, with edges fringed by mycelium formation. After 2 weeks, parts of the mycelia turned dark. On Blakeslee's malt extract agar half of the colony and some of the my- celia were dark black-green. On corn-meal agar abundant true mycelia were produced. Carbon assimilation, nitrogen assimilation, fermentation and vita- min requirements were typical of the species (De Hoog, 1983) (data not presented).

The P. syringae isolate was a Gram-negative rod (Fig. ld) , and motile by 1-4 lophotrichous polar flagella. Colonies on nutrient agar and trypticase agar were slightly irregular with an entire edge. On ATCC medium No. 73, colonies were smooth and mucoid. We did not perform pathogenicity tests on this iso- late. The P. cepacia isolate was isolated and described by Spurr and Knudsen (1985).

In a comparison of the 4 top antagonists on infection by the 2 PeniciUium spp. (Figs. 2 and 3), all demonstrated significant control compared with the check. Pseudornonas cepacia provided the best protection against both P. dig-

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DAYS AFTER I N O C U L A T I O N

Fig. 2. Effect of antagonists on grapefruit infection by P. italicum. Each treatment involved 5 fruit with 2 wounds per isolate. Experiments were repeated 4 times and the data pooled. Treatments with the same letters do not differ significantly (P < 0.05) according to Dunan's multiple range test,

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itatum and P. italicum. Although D. hansenii, A. puUulans and P. syringae ex- hibited antagonism equal to P. cepacia for the first week of lesion development, they were not as effective after 13 days against P. italicum (Fig. 2 ). There was a tendency for antagonism by the two bacteria to be expressed over a longer period than by the two yeasts.

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DISCUSSION

All four antagonists identified in this study have potential as biocontrol agents against PeniciUium rots of citrus. Their effectiveness can probably be enhanced by manipulating their preparation and method of application.

Pseudomonas cepacia is an effective biocontrol agent on plant surfaces (Jan- isiewicz and Roitman, 1988), and against soil pathogens (Spurr and Knudsen, 1985). It has also been reported as a plant pathogen (Cother and Dowling, 1985). A variety of antibiotic compounds has been identified from strains of P. cepacia. Although P. cepacia appears to control PeniciUium rots by anti- biosis, its application to the peel may not result in the introduction of anti- biotics into the food chain when fruit are sold on the fresh market.

Pseudomonas syringae is a common plant pathogen that has been divided into over 40 pathovars based on host range. Pseudomonas syringae is found commonly on plant surfaces where it does not cause disease (Blakeman and Fokkema, 1982). It also is an effective antagonist of Penicillium expansum Thorn. rot of apples (Janisiewicz, 1987). Use of antagonistic strains of P. syr- ingae on citrus, where it does not cause disease, would seem warranted. How- ever, implied guilt through association with other pathogens may restrict de- velopment of this antagonist commercially.

Aureobasidium pullulans is one of the most universally distributed micro- organisms in the ecosystem. It is widely distributed on plant surfaces where it can function as a biocontrol agent (Fokkema and Lorbeer, 1974). A weak par- asitic relationship has been reported for A. puUulans on grape leaves (Vercesi et al., 1982).

Debaryomyces hansenii is a common organism occurring in milk and cheese (Rij, 1984). It has not been reported previously as a biocontrol agent against plant pathogens. Interestingly, D. hansenii was suggested as a biocontrol agent in cheese to reduce Clostridium populations which cause gas-pocket defects (Fatichenti et al., 1983). Since D. hansenii or its relatives are not pathogenic to plants, and appear to control Penicillium rot pathogens without antibiotic production, it is probably the best prospect among the antagonists identified to gain acceptance commercially. Also, since it is a species that is consumed commonly in milk and cheese, potential toxicity to humans should not be a problem.

ACKNOWLEDGMENTS

We thank Dr. Roy E. McDonald, USDA Horticultural Research Laboratory, Orlando, FL for providing the citrus used in these studies and Dr. Michael E. Wisniewski, USDA ARS Appalachian Fruit Research Station for help in ana- lyzing and presenting the data. This work reflects the excellent technical as- sistance of Brian E. Otto, J.D. Franklin and Wilbur L. Hershberger.

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T h i s r e s e a r c h was s u p p o r t e d by a g r a n t ( U S - 1 0 1 9 - 8 5 ) f r o m the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l Agr i c u l t u r a l R e s e a r c h a n d D e v e l o p m e n t F u n d ( B A R D ) .

M e n t i o n of a t r a d e m a r k , w a r r a n t y , p r o p r i e t a r y p r o d u c t , or v e n d o r does n o t c o n s t i t u t e a g u a r a n t e e by t he U.S. D e p a r t m e n t o f A g r i c u l t u r e a n d does n o t i m p l y its a p p r o v a l to t he exc lus ion o f o t h e r p r o d u c t s or v e n d o r s t h a t m a y be sui table .

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