Antonie van Leeuwenhoek63: 23-27, 1993. © 1993KluwerAcademicPublishers. Printedin the Netherlands.

Mycelial elongation and sporulation of two fungi on amended media in light or dark

V.M. Russo 1 & A.J. Pappelis 2 1 United States Department of Agriculture, South CentralAgricultural Research Laboratory, POB 159, Lane, OK 74555, USA; 2Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901, USA Receivedand accepted 19 October 1992

Key words: 6-benzyladenine, Botrytis allii, caffeic acid, chlorogenic acid, Colletotrichum dematium, kinetin Abstract. Botrytis allii and Collectotrichum dematium are onion pathogens which can infect in the field and

cause decay in storage. Some phenolics can hinder development of these fungi, but the effect of cytokinins is not clear. Cytokinins (kinetin or 6-benzyladenine) or phenolics (caffeic or chlorogenic acids) were added to agar at concentrations of 0 to 10-3M. Cultures were continuously irradiated with fluorescent light or maintained in the dark for 6 days. On unamended media, final mycelial elongation was 45 or 17.8 mm and sporulation was 28 or 10.6 x 104 spores/ml for Botrytis and Colletotrichum, respectively. For Botrytis, mycelial elongation was slightly (5%) but significantly increased and sporulation increased by 21% by incubation on phenolics as compared to cytokinins. Mycelial extension of Colletotrichum was not affected by amendment. Sporulation of Colletotrichum on kinetin was 16 to 28% greater than on the other amendments. As amendments concentration increased elongation of mycelia of both fungi decreased. Sporulation of Botrytis increased by 60% as amendment concentration increased from 0 to 10-5 M and then decreased 25% at 10-3 M. As amendment concentration increased from 0 to 10 -3 M, sporulation of Colletotrichum increased by 45%. Incubation in light increased mycelial extension 3 to 17% for Botrytis and Colletotrichum respectively, and sporulation was increased approximately 78% for both fungi. These compounds do not appear to inhibit development of these Botrytis or Colletotrichum species in culture.

Introduction

Fungal morphogenesis is affected by naturally occurring chemicals found in plants and/or fungi (Turian 1974; Schonbeck & Schlosser 1976). One group, phenols, are often implicated in host resistance to disease. Walker et al. (1929) described the first case of disease resistance due to a phenol, protocatechuic acid. Walker & Stahmann (1955) reported that catechuic and protocatechuic acids are the principal phenols in onion responsible for protection against some fungal pathogens. Depending on bulb scale color, Somesekhara (1974)

found that exposing onion bulb scales to ambient air increased levels of chlorogenic and caffeic acids up to 0.01 and 1.8 mg g-l, respectively. Caffeic and chlorogenic acids polymerize to lignin-like compounds and act as nonspecific inhibitors of many fungal enzymes. The quinone derivatives of these phenols may be responsible for the hypersensitive response (Kud 1976). Somesekhara (1974) also reported that caffeic acid at concentrations above 10-7M stimulated germination of Botrytis allii Munn. conidia, but chlorogenic acid at 10-5 to 10 -7 M inhibited germination between pH 4 and 6. Fiussello et al. (1975) suggested that B. cinerea

24 Pers. ex Fr., has lignin-degrading activity, and it may be that some fungi can use these compounds as substrates. Plant hormones and their precursors may maintain host cells in a condition which may affect the development of the hypersensitive response (Elstner 1983; Vidhyasekaran 1988). Cytokinins, produced by certain fungi influence host susceptibility to fungal invasion (Miller 1967; Sequeira 1973). Cytokinins, above 3ppm, can inhibit growth, increase protein content, but reduce hydrolase synthesis of Verticillium albo-atrum Reinke & Berth (Patil & Dimond 1968). Roten et al. (1978) stated that continuous illumination with light in the visible portion of the spectrum causes a sporulation-inhibiting enzyme to accumulate in many fungi. Tan & Epton (1973) reported that dark incubation retarded sporulation of B. cinerea, but had little effect on mycelial elongation. Majumbar & Sen (1975) found that for several Colletotrichum spp. isolates little or no sporulation occurred on colonies maintained in the dark. Sporulation in some fungi has been hypothesized to be controlled by a yet to be isolated photoreceptor system termed mycochrome (Yamamura et al. 1978). This study was undertaken to determine if mycelial elongation and sporulation of two onion pathogens are affected by selected cytokinins or phenols, and if effects are mediated by dark vs light incubation.

Materials and methods Cytokinins [Kinetin (6-(Furfurylamino) purine); 6benzyladenine], or phenols [caffeic acid (4-dihydroxycinnamic acid); chlorogenic acid (3-caffeoyl quinnic acid)], were dissolved in warm sterile distilled water, and added to 2% agar Czapex-Dox medium after autoclaving and cooling. Final concentrations were 10 -7, 10 -5, and 10-3 M. Controls were agar to which sterile water only was added. Ten ml of amended or unamended media were dispensed into plastic Petri-dishes (60 x 15 mm). Fungi were isolated from diseased onions. Small amounts of spores and mycelia of B. allii Munn. (Moniliales) (BA), and C. dematium Berk. (Mela-

nconiales) (CD), were taken by sterile transfer needle from 14-day-old cultures and placed at the extreme edge of media in Petri-dishes. Cultures were incubated approximately 46cm below coolwhite fluorescent light (Sylvannia, 350-750nm, 8800 ergs cm -2 S-1) at 24 + I°C for six days. Alternatively, inoculated Petri-dishes were loosely wrapped with aluminum foil and placed in a closed cabinet in the same room for the same length of time. The experimental design consisted of three replicates per treatment combination (2 fungi x 2 incubation regimes (light vs dark)x 4 amendments x 4 amendment concentrations (0, 10 -7, 10-5, and 10-3 M)). There was always an unamended control plate for each treatment combination. The experiment was repeated. Mycelial elongation along each of two radii, separated by an angle of 45 °, was measured from the point of inoculation and averaged. Each culture was flooded with 10 ml of sterile distilled water. Flooding was followed by gentle agitation with a camel hair brush to dislodge conidia and suspended spores were decanted into 25 ml beakers. Aliquots (0.33ml) of spore suspensions were placed in a clean glass vial and brought to 5 ml with distilled water. Vials were shaken for several seconds and a 0.1 ml aliquot from each vial was placed on a Howard Mould Counting chamber. The number of conidia in each of three randomly chosen fields (400 x total magnification) were counted and converted to number of spores/ml. Data was analyzed by the general linear models procedures in SAS (SAS, Inc., Cary, N.C.).

Results Fungal responses to incubation in fluorescent light or in the dark, and by presence and concentration of amendments are in Table 1. For both fungi, incubation in light significantly increased mycelial elongation and sporulation over dark cultured colonies. Mycelial elongation and sporulation of BA colonies incubated on agar amended with caffeic and chlorogenic acids were significantly higher than those on kinetin or 6-benzyladenine amended agar. For BA, although significant, the increases

25 were only 5%, and the biological importance of that amount of difference is uncertain. Mycelial elongation for CD colonies did not differ significantly among amendments. Standard error of the means for spore numbers were approximately 8.1% (range 4-10%) of means for BA and 7.6% (range 4-9%) of means for CD. On kinetin amended agar, CD had the highest sporulation, while sporulation on all other amended media were all less than for kinetin. Mycelial elongation for both fungi decreased in a linear manner as concentration of amendments increased. Sporulation of BA was distributed in a quadratic fashion, increasing from no amendment (control) to 10-5 M before decreasing at 10-3 M. For CD, sporulation increased significantly and linearly with increasing concentration of amendments. A zone of discoloration was formed only at the edge of CD colonies grown on medium amended with the highest concentration (10 -3 M) of phenols but not cytokinins.

Discussion

Many fungal responses are stimulated by light and it is well known that certain wavelengths affect sporulation and growth. Both BA and CD appear to respond in a similar manner as B. cinerea, to incubation in light (Tan & Epton 1973). The lack of interaction between light or dark incubation and amendment occurrence or concentration suggest that activity of cytokinins or phenols are not affected by continued exposure to a multi-wavelength light. Additional research is needed to determine if specific wavelengths will affect fungal sporulation or change the activity of the compounds tested. Harvey et al. (1971) reported that kinetin did not affect growth of Cronartium ribicola A. Fisch. For BA, one or both cytokinins reduced mycelial elongation slightly but did affect mycelial elongation of CD when comparison was made to phenolics. In higher plants, division of specialized somatic cells is mediated by cytokinins (Galston 1950). Mediation

Table 1. Effect of light during incubation, m e d i u m a m e n d m e n t , and concentration of a m e n d m e n t on mycelial elongation and sporulation of B. allii and C. dematium. B. allii Treatments

C. dematium

Mycelial elongation

Sporulation

Mycelial elongation

Sporulation

(mm)

( x 104 m1-1) (ram)

( x 104 m1-1)

41.4 40.3 ,1

42.8 9.4 ••

17.4 14.6 ••

16.3 3.6 ,•

42.2a 2 42.2a 40.1b 40. lb

41.5a 39. la 30.6b 33.4b

17.0a 17.0a 16.9a 16.6a

14.0b 12. lb 16.7a 12.5b

45.0 41.2 40.7 37.8 **, - L

28.0 38.1 44.9 33.6 *, Q

17.8 16.7 16.8 16.1 **, - L

10.6 15.3 13.4 15.9 **, L

Fluorescent light (F) Yes No Significance

Amendment (A ) Chlorogenic acid Caffeic acid Kinetin 6-benzyladenine

Concentration (C) 03 10 -7 10 -5 10 -3 Linear significance

ins, *, ** = non-significant, or significant at the 5% or 1% level respectively; L = linear, Q = quadratic. None of the interactions were significant. 2Numbers in a column followed by the same letter are not significantly different, D M R T (p = 0.05). 30 = u n a m e n d e d control; 10 7, 10-5, or 10 3 M of the a m e n d m e n t s .

26 of cell division resulting in elongation of mycelia is likely not controlled by cytokinins. As concentration increased, mycelial elongation decreased by upto 16%. However, sporulation generally increased as concentration increased. This would suggest that either both groups of amendments were being used as energy sources, or that phenols were used as an energy source and the cytokinins were stimulating cell division. Phenolics are produced in response to stress, including infection by fungi. These compounds precipitate proteins in plant cells and produce an environment that is incompatible with fungal growth (Ku6 1972). A review by Vidhyasekaran (1988) stated that phenolics decrease production of, or inactivate fungal enzymes or detoxify fungal toxins. Catechol does not affect conidial germination or mycelial growth of F. oxysporum f. sp. lycopersici (Schlecht) Sacc., but may suppress its toxin production (Chet et al. 1978). Only the quinone form of chlorogenic acid inhibited spore germination of V. albo-atrum (Patil et al. 1964). Caffeic acid inhibited germination of spores of Diplodia zeae (Schw.) Lev. (Dabler et al. 1969). The discoloration seen in advance of the mycelia of CD may be due to phenols interacting with fungal metabolites (Wenrman & Swain 1953). This interaction may represent an alteration of the phenols to a form with a different toxicity. Phenols are generally considered to be fungitoxic (Schonbeck & Schlosser 1976). However, Lee & LeTourneau (1958) reported that phenols can serve as energy sources for V. albo-atrum. Miller et al. (1991) suggested that caffeic acid increases the activity, and presumably the virulence, of Fusarium oxysporum (Schlecht) f. sp. asparagi (Cohen). Walker & Stahmann (1955) reported that BA is inhibited by catechuic and protocatechuic acids. Somasekhara (1974) reported that caffeic and chlorogenic acids levels are increased in onions exposed to air. These phenolics in concentrations equivalent to those reported by Somasekhara (1974) did not appear to play a major role in the development of BA or CD. Because mycelial elongation is only slightly affected by caffeic and chlorogenic acids, it may be that these compounds would not impede attempted colonization of onion by BA or CD.

Further research is needed to determine the role of these compounds in onion. Caffeic and chlorogenic acids are implicated in initiating the hypersensitive response (Ku6 1972), a condition in which the cell environment, not necessarily the individual phenol, becomes toxic to the invading fungus. Outside of the cell, caffeic and chlorogenic acids may not be toxic to BA or CD.

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