Effects of Bioregulators on Growth and Toxin Formation in Fungi

13 Effects of Bioregulators on Growth and Toxin Formation in Fungi F R E D E R I C K W. PARRISH

Downloaded via UNIV OF ARIZONA on July 22, 2018 at 08:22:58 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, L A 70179 S U S A N B. JONES Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

In stationary culture on media containing glucose or sucrose as carbon source, growth of some a f l a t o x i n -producing s t r a i n s of Aspergillus flavus and A. p a r a s i t i c u s was not affected by PIX bioregulator, but production of aflatoxins was i n h i b i t e d . Formation of citrinin by a s t r a i n of P e n i c i l l i u m citrinin was inhibited by the addition of PIX to yeast extract -sucrose medium or r i c e grain. No e f f e c t of PIX was observed on the formation of secalonic acid D by a s t r a i n of P e n i c i l l i u m oxalicum growing on yeast extract-sucrose medium or r i c e grain. The formation of penicillic acid by a s t r a i n of Aspergillus sulfureus growing on the sucrose-based medium was s l i g h t l y i n h i b i t e d in the presence of PIX. Transmission electron microscopy showed larger c e l l s with thicker cell walls in the PIX-treated mycelium compared to an untreated c o n t r o l . Differences were also apparent in the lipid reserves and mitochondria.

Bioregulators play an important r o l e in agriculture because of t h e i r e f f e c t s on functions such as root development, flowering, e t c . The e f f e c t s of man-made, agrichemical bioregulators have been concerned mainly with growth, y i e l d , and composition of a wide variety of plant species (1,2). Few studies have been reported on the e f f e c t s of bioregulators on microorganisms. Erwin and coworkers Ο,Α) reported that growth retardants, including N,N-dimethyl piperidinium chloride (PIX - BASF), when applied to C a l i f o r n i a cotton, reduced the severity of V e r t i c i I l i u m w i l t symptoms. However, the main reason for applying PIX to cotton is to decrease growth in the upper region of the plant to produce a more compact c o n i c a l form. This allows the cotton plants to be spaced closer together giving greater y i e l d . PIX has also been used to shorten the height of winter wheat thereby reducing lodging and increasing the y i e l d (5). B e n e f i c i a l e f f e c t s of a p p l i c a t i o n of PIX have also been found with c i t r u s , grapes, potatoes, and onions (2).

This chapter not subject to U.S. copyright. Published 1984, American Chemical Society

Ory and Rittig; Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

142

BIOREGULATORS: CHEMISTRY AND USES

Our interest in PIX stemmed from studies of t h i s bioregulator on sorghum performed by Dr. Harold Gausman (USDA, Lubbock, TX) and h i s colleagues at Weslaco, TX. Applications of PIX at a rate of 1.4 oz/acre caused a s t a t i s t i c a l l y s i g n i f i c a n t increase in the protein content of sorghum (11.88% to 12.47%), and a concomitant decrease in starch content (74.47% to 73.48%). Amino acid analysis showed no differences between the grains from treated and control plants. This raised the question as to whether addition of PIX to culture media would increase the production of c e l l u l a s e enzymes by Trichoderma reesei. The n a t u r a l l y occurring bioregulators, indole-3-acetic a c i d and g i b b e r e l l i c a c i d , at concentrations of lO',M, double the production of protein and c e l l u l a s e with Trichoderma species grown on carboxy methyl c e l l u l o s e (6). Therefore, the e f f e c t of PIX on T. reesei was investigated. EFFECT OF PIX ON TRICHODERMA REESEI Commercial PIX solution was s t e r i l i z e d by f i l t r a t i o n through a O.45 micron M i l l i p o r e f i l t e r and added to h e a t - s t e r i l i z e d , standard Trichoderma medium (7) containing O.5% carboxy methyl c e l l u l o s e . The f i n a l concentration of PIX was 1 mg/ml (6.68 mM). After inoculation with Trichoderma reesei QM 6a spores, the culture media were shaken for 10 days at 28 C. The control medium, with no addition of PIX, produced c e l l u l a s e enzyme, but no growth of the Trichoderma occurred in the medium containing PIX. When PIX was added to the culture medium 2 days a f t e r inoculation, growth was arrested. Incidentally, ear and kernel rot of corn by Trichoderma reesei has been reported recently (8), and i t would be of interest to examine the e f f e c t of PIX on t h i s disease in corn. FUNGAL TOXINS A serious problem involving fungi and animal feed was encountered in 1960, when 100,000 young turkeys died in the course of a few months (9)· The disease proved to be non-infectious, and was eventually recognized as being caused by aflatoxins produced by Aspergillus flavus or Aspergillus p a r a s i t i c u s growing on animal feed. The toxins can be produced by these fungi in corn, cottonseed, peanuts, and other commodities, and they a f f e c t pigs and calves in addition to poultry. In the years since 1960, the e f f o r t s of many s c i e n t i s t s have been directed to various aspects of the a f l a t o x i n problem r e s u l t i n g in thousands of published papers. Despite these e f f o r t s , serious problems of aflatoxins in feed remain. At Southern Regional Research Center, studies of fungal toxins comprise a major program. Some of the toxins being studied, and the fungi responsible for t h e i r formation, are shown in Table I. The fungi which produce these toxins are plant pathogens and postharvest saprophytes in the c l a s s Hyphomycetes. Major emphasis is on the four aflatoxins from Aspergillus flavus and Aspergillus p a r a s i t i c u s .


13.

Growth and Toxin Formation in Fungi

PARRISH AND JONES

TABLE I.

143

Fungal Toxins from some A s p e r g i l l i and P e n i c i l l i a

Toxin

Source

Aflatoxins

Aspergillus flavus Aspergillus p a r a s i t i c u s

Citrinin

Pénicillium citrinum Pénicillium viridicatum

P e n i c i l l i c Acid

Pénicillium spp. Aspergillus spp.

Secalonic Acid D

Pénicillium oxalicum

AFLATOXINS Culture conditions can influence the extent of a f l a t o x i n production. For example, y i e l d s of a f l a t o x i n are greater when A s p e r g i l l i are grown on glucose/ ammonium n i t r a t e medium for 7 days at 25°C. or 29°C. than on Czapek's medium, which is based on the same concentration of glucose (10). With a semisynthetic medium based on 2% yeast extract and 20% sucrose, some A s p e r g i l l i which produce aflatoxins in s t i l l culture do not do so in shake culture. Liquid media rather than s o l i d media were chosen for most of these experiments because fungal growth is generally slower on s o l i d media, and can lead to decreased l e v e l s of mycotoxin production. In addition, i s o l a t i o n of fungal toxins from s o l i d media, e.g., corn and peanuts, often requires additional steps to remove constituents extracted from the a g r i c u l t u r a l commodity, e.g., o i l s and f a t s , which would otherwise i n t e r f e r e with the chromatographic separation and determination of the toxins. Temperature, moisture content, and surface area of substrates have been shown to be important factors in fungal growth and a f l a t o x i n formation (11,12). The amounts formed on l i q u i d media at 25°C. are often 150-200 times greater than the amounts formed a t 15°C. For studies of a f l a t o x i n formation, glucose/ammonium n i t r a t e was selected as the synthetic medium (13) and yeast extract (2%)/sucrose (20%) as the semisynthetic medium (14). Fungi were grown for 6 days a t 29 C.inshake or s t a t i c cultures. No adjustment of the i n i t i a l pH o f the medium was made. Experiments were replicated three times. At the end of the 6-day growth period, a volume of chloroform equal to that of the c u l t u r e medium was added to each f l a s k . The f l a s k anditscontents were heated and magnetically s t i r r e d u n t i l the chloroform vapor reached the mouth of the f l a s k . The mixture was cooled to room'temperature then f i l t e r e d through a sintered glass funnel. The mycelium was washed with chloroform, and the washings were added to the f i l t r a t e . The weight of mycelium was determined by drying the mycelial mats f o r 24 hr a t 70 C. The f i l t r a t e and washings were transferred to a separatory funnel, and a f t e r shaking, the chloroform layer was removed. The aqueous layer was re-extracted with an equal volume of chloroform. These extractions were S u f f i c i e n t to remove the aflatoxins from the e

e



144

mycelium and the culture medium. The combined chloroform extracts were evaporated to dryness at 40 C in vacuum (15). The residue which remained after evaporation of the chloroform was dissolved in a known volume of chloroform and aliquots were applied to a 20 cm χ 20 cm thin-layer plate of s i l i c a gel together with known amounts of a f l a t o x i n B l , B2, G l , and G2 standards. The plate was developed for 40 min in ether followed by 20 min in ether/methanol/water (96/3/1). The amounts of the i n d i v i d u a l aflatoxins were determined by measuring the fluorescence with a spectrodensitometer connected to a data handling system. The r e p r o d u c i b i l i t y of the assay was ± 6 % . In shake culture on glucose/ammonium n i t r a t e medium, Aspergillus p a r a s i t i c u s NRRL 2999 produced aflatoxins in the control medium, but none in the presence of 50 pg/ml of PIX. When PIX was added at the time the culture media were inoculated with fungus spores (ca. O.5 χ 10 spores), no difference in mycelial weight was found between the control and PIX-treated cultures. The same r e s u l t was observed when addition of PIX was made 24 hr after addition of dormant spores to the culture medium. By t h i s time the dormant spores had undergone transformation to the vegetative mycelial stage. These r e s u l t s show that the e f f e c t of PIX is not due simply to prevention of swelling and germination of dormant spores or to an e f f e c t on the extent of mycelial growth, as has been observed with so many other i n h i b i t o r s of a f l a t o x i n formation. The same e f f e c t s of PIX were also observed when the concentration of PIX in the culture media was increased to 400 or 4000 pg/ml. These higher l e v e l s of PIX were tested because Bennett and coworkers (16) found a concentration dependence of acetone or ethanol on a f l a t o x i n formation. They found in a glucose-containing resting c e l l medium, which lacks a nitrogen source and allows a f l a t o x i n synthesis, that Aspergillus parasiticus NRRL A-16,462 was markedly i n h i b i t e d (99%) in a f l a t o x i n formation by the presence of 1 M acetone, but with O.01 M or O.1 M acetone, a f l a t o x i n formation was stimulated 2.3-fold. Similar r e s u l t s were observed at the same concentrations of ethanol; 95% i n h i b i t i o n at 1 M, and 1.6-fold stimulation at O.01 or O.1 M. Identical results for the e f f e c t of PIX on growth and a f l a t o x i n formation were found with Aspergillus flavus 1000-A when PIX was added at 0, 24, or 48 hr a f t e r inoculation of the culture medium. In addition, on yeast extract/ sucrose medium in s t a t i c culture, a f l a t o x i n formation by Aspergillus p a r a s i t i c u s NRRL 2999 was i n h i b i t e d by PIX. However, in shake culture, t h i s organism did not produce aflatoxins in the control medium which had no PIX addition. Similar r e s u l t s of i n h i b i t i o n of a f l a t o x i n formation by PIX were found on glucose/ammonium n i t r a t e medium in shake or s t a t i c culture with Aspergillus parasiticus SU-42, with two other s t r a i n s of Aspergillus flavus (SRRC 31 and SRRC 37), and another s t r a i n of Aspergillus p a r a s i t i c u s (SRRC 235) in s t a t i c culture on yeast extract/sucrose medium. I t is noteworthy that, when the PIX-containing medium was heats t e r i l i z e d , the i n h i b i t o r y e f f e c t of PIX on a f l a t o x i n formation was not observed. e


13.

PARRISH AND JONES


145

INHIBITORS OF AFLATOXIN FORMATION A wide variety of chemicals have been shown to i n h i b i t a f l a t o x i n synthesis on synthetic, semisynthetic or s o l i d media o f a g r i c u l t u r a l commodities. The l i s t includes p-aminobenzoic acid, sulfanilamide, a n t h r a n i l i c acid, potassium s u l f i t e , and potassium f l u o r i d e (17); mercaptoethanol (18); c a f f e i n e (19); the organophosphate i n s e c t i c i d e s NALED and DICHLORVOS (20-23); and unidentified compounds in a chloroform extract of carrot roots (24). Many of these i n h i b i t o r s , e.g., p-aminobenzoic acid, also markedly i n h i b i t fungal growth in a p a r a l l e l fashion. However, DICHLORVOS, mercaptoethanol, and PIX, which i n h i b i t a f l a t o x i n formation at s i m i l a r concentrations (Table I I ) , do not strongly i n h i b i t fungal growth. TABLE I I . Comparison of i n h i b i t i o n of a f l a t o x i n formation and mycelial growth by DICHLORVOS, mercaptoethanol, and PIX

Compound

Cone, (pg/ml)

% Inhibition Growth Aflatoxin

100

99

29

Mercaptoethanol

80

97

30

PIX

50

100

DICHLORVOS

0

A possible explanation of " i n h i b i t i o n " of a f l a t o x i n production which needs to be considered for DICHLORVOS, mercaptoethanol, and PIX, is that in the presence of these compounds the aflatoxins are degraded. Degradation of aflatoxins by mycelia of A s p e r g i l l i (25) and by potassium b i s u l f i t e (26) has been reported. If the mechanism of action of PIX is i n h i b i t i o n of a f l a t o x i n biosynthesis, i t would be possible to investigate the enzymes and intermediates that are affected by following the approaches described by Bennett and Lee (27). Aflatoxins are produced by a polyketide pathway wherein the carbon skeleton is formed from acetate, with the methoxy methyl group being derived from methionine (28,29). Methods available to study biosynthetic pathways include the use of l a b e l l e d precursors, blocked mutants, metabolic i n h i b i t o r s , and c e l l - f r e e systems (27). TRANSMISSION ELECTRON MICROSCOPY Another approach to an understanding of the behavior of fungi with respect to production of metabolites is to examine the fungi by microscopy in order to observe morphological and u l t r a s t r u c t u r a l differences. Photomicrographs and transmission electron micrographs of Aspergillus p a r a s i t i c u s NRRL 2999 grown f o r 6 days a t 27C.on yeast extract/ sucrose medium were obtained. The mycelia were fixed in 2% glutaraldehyde in O.05 M sodium cacodylate buffer (pH 7.0) f o r 5 hr at 2 5 , , and post-stained in 1% osmium tetroxide in the same buffer. Fixed samples were dehydrated through graded aqueous acetone,



146

including en bloc staining in 2% uranyl acetate/ 70% acetone for 1 hr, and embedded in Spurr low-viscosity r e s i n . Thin sections were stained with aqueous uranyl acetate and lead c i t r a t e and observed in a Zeiss EM 10 at 60 kV. The transmission electron micrographs are shown in Figures 1,2. Control mycelium from shake culture (Figure l a ) , which produces no a f l a t o x i n s , showed a densely-staining cytoplasm with many round l i p i d inclusions. The organelles do not stand out, and no e x t r a c e l l u l a r , f i b r i l l a r material is present. Control mycelium from s t a t i c culture (Figure l b ) , which produces a f l a t o x i n s , was d i f f e r e n t from the control shake mycelium (Figure l a ) . The c e l l s appear to be a c t i v e l y metabolizing, and the mitochondria look well-formed. The l i p i d inclusions were modified so that they have dark material at boundaries, which may be l i p i d soluble product. The l i p i d reserves were not great, and there was a l o t of apparent a c t i v i t y in membrane-bound organelles of both dégradâtive and synthetic types. Mycelium produced in s t a t i c culture in the presence of s u f f i c i e n t p-aminobenzoic acid to i n h i b i t a f l a t o x i n formation (Figure 2a), was severely affected by p-aminobenzoic acid, and i t was d i f f i c u l t to f i n d a l i v e c e l l to photograph. The u l t r a s t r u c t u r e was i n d i s t i n c t , the mitochondria were poorly developed, and there were no l i p i d reserves. Mycelium from the PIX-treated fungus (Figure 2b), which d i d not produce a f l a t o x i n s , showed c e l l s which were larger than the other 3 samples and the c e l l walls were thick. There were considerable l i p i d reserves and some black inclusions, possibly pigments, within vacuoles. There appeared to be a low l e v e l of dégradâtive/synthetic a c t i v i t y of the same type as was observed with the c o n t r o l , s t a t i c mycelium. The mitochondria were also d i f f e r e n t from the control mitochondria. Those from the PIX treatment have densely-staining matrix and t i g h t l y packed c r i s t a e ( i n t e r i o r laminations) and occur in b i z a r r e shapes. The implications of these observations with respect to a c t i v e phosphorylation is s t i l l under investigation. CITRININ C i t r i n i n is a nephrotoxin which can a f f e c t swine. Pénicillium citrinum 5927 was grown in s t a t i c culture on yeast extract/sucrose medium or on r i c e grains with 50% added water. The r i c e culture medium was a c i d i f i e d to pH 1.5 by addition of 6 M hydrochloric a c i d , and was extracted twice with chloroform. The chloroform layer was extracted with O.1 M sodium bicarbonate to remove citrinin, and a f t e r a c i d i f i c a t i o n the citrinin was extracted into chloroform. This extraction procedure gave a sample s u i t a b l e for t h i n layer chromatography. The sodium bicarbonate treatment was not necessary for the chloroform extract of a c i d i f i e d yeast extract/sucrose medium in order to produce a sample for TLC. The assay for citrinin was performed by Stubblefield's procedure (30) f o r TLC on s i l i c a plates containing EDTA. Following development in benzene/acetic acid (95/5), the d r i e d plates were scanned on a spectrodensitometer at an e x c i t a t i o n wavelength of 365 nm and emission wavelength of 505 nm. Peak areas were calculated using an automated data system. C i t r i n i n formation was inhibited by PIX at the only concentration l e v e l tested, 3.4 mg/ml in yeast extract/sucrose medium and 17 mg/gram of r i c e .


13.

PARRISH AND JONES


147

F i g u r e 1. A s p e r g i l l u s p a r a s i t i c u s NRRL 2999 grown in y e a s t e x t r a c t / s u c r o s e l i q u i d medium f o r 6 days a t 27 °C. Key: a — shake c u l t u r e , 135 rpm; no a f l a t o x i n s produced; b — s t i l l c u l t u r e , a f l a t o x i n s produced, χ 10,000.

American Chemlctf Society Library 1155 16th St. N. W. Ory and Rittig; Washington, D. Bioregulators C. 20038 ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

148


F i g u r e 2. A s p e r g i l l u s p a r a s i t i c u s NRRL 2999 grown in s t i l l c u l t u r e s w i t h i n h i b i t o r s o f a f l a t o x i n p r o d u c t i o n . Key: a — p-aminobenzoic a c i d (PABA), 8 mg/ml, added a t 0 time; b — Ν,Ν-dimethylpiperidine c h l o r i d e ( P I X ) , 50 μg/ml, added a t 0 t i m e , χ 10,000. Legend: M, m i t o c h n o d r i o n ; L, l i p i d i n c l u s i o n ; N, n u c l e u s ; V, v a c u o l e ; W, c e l l w a l l ; f , e x t r a c e l l u l a r f i b r o u s material.


13.

PARRISH AND JONES


149

SECALONIC ACID D A toxin from 5 s t r a i n s of Pénicillium oxalicum was isolated and i d e n t i f i e d as secalonic acid D by Steyn (31). Extracts of t h i s fungus, which commonly i n f e c t s corn, are toxic to laboratory and domestic animals. Pénicillium oxalicum 5209 was inoculated into yeast extract/sucrose medium (25 ml) or r i c e grains (5 g) with 50% added water in s t a t i c culture at 28*C. The e f f e c t of PIX was tested at 3.4 mg/ml in yeast extract/sucrose medium and at 17 mg/gram of r i c e . Ciegler and coworkers (32) showed that secalonic acid D production with t h i s organism on yeast extract/sucrose medium or on corn was greater in s t a t i c culture than in shake culture. These authors found l e v e l s of secalonic acid D a f t e r 14 days inoculation to be about 2 mg/ml on yeast extract/sucrose medium and about 800 mgAg on r i c e . Using the same extractant as C i e g l e r , i . e . , methylene chloride, the secalonic acid D was assayed by the high performance l i q u i d chromatographic procedure of Reddy e t a l (33). This involved removing the methylene c h l o r i d e by evaporation, d i s s o l v i n g the sample in a c e t o n i t r i l e , and i n j e c t i n g i t on to a uBondapakC.-18reverse phase column. The eluant was acetonitrile/water/acetic acid/tetrahydrofuran (4/3/O.5/O.5) in the i s o c r a t i c mode with detection at 365 nm. Other compounds with s i m i l a r retention times to secalonic acid D interfered with the assay. However, these i n t e r f e r i n g compounds were removed r e a d i l y by extracting the a c e t o n i t r i l e solution, containing secalonic acid D, with hexane. Secalonic acid D c r y s t a l l i z e d r e a d i l y on standing in the a c e t o n i t r i l e s o l u t i o n . I t s i d e n t i t y was confirmed by its absorbance c h a r a c t e r i s t i c s ( max 339 nm) as reported by Reddy e t a l (33), and by mass spectral comparison with authentic material giving the 638 molecular ion and other fragmentation ions. We found s i m i l a r l e v e l s of secalonic acid D formation on yeast e x t r a c t / sucrose medium and on r i c e to those found by Ciegler e t a l (32). However, there was no i n h i b i t i o n of secalonic acid D formation by PIX. PENICILLIC ACID FORMATION P e n i c i l l i c acid is a compound produced by a number of P e n i c i l l i a and A s p e r g i l l i which induces f a t t y l i v e r degeneration in q u a i l and l i v e r c e l l necrosis in mice (34). A s p e r g i l l u s sulfureus 97 was grown on yeast extract/sucrose medium for 13 days at 28°C. in shake or s t a t i c culture. Work-up was performed as described by Ciegler e t a l (34). The a c i d i f i e d (pH 1.5) medium was extracted with chloroform, and the extracts were assayed by t h i n layer chromatography on s i l i c a g e l with chloroform/ ethyl acetate/formic acid (60/40/1) as developing solvent. Exposure of the a i r - d r i e d p l a t e to concentrated ammonia gave a blue fluorescent d e r i v a t i v e having Rf O.5. The e f f e c t of PIX addition on p e n i c i l l i c acid formation was to cause 32% i n h i b i t i o n in the presence of 2.4 mg/ml of PIX. FUTURE WORK The survey of the e f f e c t s of PIX on a number of other fungal toxins is continuing with emphasis on the mechanism of PIX action in


150


i n h i b i t i n g a f l a t o x i n formation. More organisms must be screened on d i f f e r e n t media, p a r t i c u l a r l y those based on cereals and oilseeds, t o determine i f the e f f e c t of PIX is a general one. ACKNOWLEDGMENTS We are grateful to George Cathey (USDA, S t o n e v i l l e , MS) f o r supplying commercial PIX; to Timothy Dobson, Beverly Maleeff, and Charlotte Thornton f o r excellent technical assistance, and to Drs. Ken E h r l i c h , Robert Ory, and Falk R i t t i g f o r h e l p f u l discussions during the course of t h i s work. The mass spectra of secalonic acid D were obtained by Michael Legendre.

LITERATURE CITED 1. 2. 3.

York, A. C. Agronomy J. 1983, 75, 663-667. BASF Technical Data Sheet. 1983. Erwin, D. C.; T s a i , S. D.; Khan, R. A. Phytopathology. 1976. 66, 106-110. 4. Erwin, D. C.; T s a i , S. D.; Khan, R. A. Phytopathology. 1979. 69, 283-287. 5. Schott, P. E. Med. Fac. Landbouww. Riyksuniv. Gent. 1979. 44, 853-865. 6. Zamost, B. L.; McLary, D. O. Biotechnology L e t t e r s . 1983. 5, 179-184. 7. Mandels, M.; Weber, J. In "Cellulases and Their Applications"; Gould, R. F., Ed.; Advances in Chemistry Series No. 95, American Chemical Society: Washington, D. C., 1969; p. 391. 8. Kumar, V.; Shetty, H. S. Current S c i . 1982. 51, 620-621. 9. Blount, W. P., J. Brit. Turkey Fed. 1961. 9, 52-61. 10. Parrish, F. W.; Wiley, B. J.; Simmons, E. G.; Long, L., J r . U. S. Army Material Command, Technical Report, Microbiology Series No. 20. 1965. 11. Northolt, M. D.; Bullerman, L. B. J. Food Protection. 1982. 45, 519-526. 12. Delucca, A. J.; Mayne, R. A.; Franz, A. O., J r . ; Ory, R. L. J. Food Protection. 1977. 40, 828-830. 13. Brian, P. W.; Dawkins, A. W.; Grove, J. F.; Hemming, H. G.; Lowe, D.;Norris, G. L. F. J. E x p t l . Bot. 1961. 12, 1-12. 14. Davis, N. D.; Diener, U. L; E l r i d g e , D. W. Appl. Microbiol. 1966. 14, 378-380. 15. Hartley, R. D.; Nesbitt, B. F.; O'Kelly, J. Nature. 1963. 198, 1056-1058. 16. Bennett, J. W.; Lee, L. S.; Gaar, G. G. Mycopathologia. 1976. 58, 9-12. 17. Davis, N. D.; Diener, U. L. Appl. M i c r o b i o l . 1967. 15, 1517-1518. 18. Gupta, S. K.; Maggon, Κ. K.; Venkitasubramanian, T. A. Appl. Environ. Microbiol. 1976. 32, 324-326. 19. Buchanan, R. L.; Hoover, D. G; Jones, S. B. Appl. Environ. M i c r o b i o l . 1983. 46, 1193-1200. 20. Rao, H. R. G.; Harein, P. K. J. Econ. Entomol. 1972. 65, 988-989.


13.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

PARRISH A N D JONES


Draughon, F. A.; Ayres, J. C. Appl. Environ. Microbiol. 1981. 41, 972-976. Bennett, J. W.; Lee, L. S.; Cucullu, A. F. Bot. Gaz. 1976. 137, 318-324. Dutton, M. F.; Anderson, M. S. J. Food Protection 1980. 43, 381-384. Batt, C.; Solberg, M.; Ceponis, M. J. Food S c i . 1980. 45, 1210-1213. Doyle, M. P.; Marth, Ε. H. Mycopathologia. 1978. 63, 145-153. Doyle, M. P.; Marth, E. H. J. Food Protection. 1978. 41, 549-555. Bennett, J. W.; Lee, L. S. J. Food Protection. 1979. 42, 805-809. B i o l l a z , M.; Buchi, G.; Milne, G. J. Amer. Chem. Soc. 1968. 90, 5017-5019. B i o l l a z , M.; Buchi, G.; Milne, G. J. Amer. Chem. Soc. 1970. 92, 1035-1043. S t u b b l e f i e l d . , R. D. J. Assoc. O f f i c . Anal. Chem. 1979. 62, 201-202. Steyn, P. S. Tetrahedron. 1970. 26, 51-57. C i e g l e r , A.; Hayes, A. W.; Vesonder, R. F. Appl. Environ. M i c r o b i o l . 1980. 39, 285-287. Reddy, C. S.; Reddy, R. V.; Hayes, A. W. J. Chromatog. 1981. 208, 17-26. C i e g l e r , A; M i n t z l a f f , H. J.; Weisleder, D.; Leistner, L. Appl. M i c r o b i o l . 1972. 24, 114-119.

RECEIVED

April 4, 1984


151

Effects of Bioregulators on Growth and Toxin Formation in Fungi

Recommend Documents